Analysis of Left Ventricular Wall Shear Stress during Diastole in Normal Subjects by Vector Flow Mapping


 Objective To observe the diastolic wall shear stress (WSS) pattern of the left ventricle (LV) by using vector flow mapping (VFM) in normal subjects. Methods A total of 371 healthy volunteers were recruited into this study and divided into four age groups. The LV WSS was measured at each diastolic phase, and the mapping of WSS was analyzed. Results Among groups I, II and III, The absolute value of WSS of Anterolateral，Inferoseptal and Anterospetal segments in phase D1；WSS values of inferolateral，Inferoseptal and Anterospetal segments in phase D4 all showed an increasing trend with age. In terms of gender differences, In most cases，women had greater diastolic WSS values compared to men. For each age group, the log-transformed WSS value appeared the increasing-decreasing-increasing trend from phase D1 to D4, with a peak value at the rapid filling phase.Multivariate backward stepwise linear regression analysis revealed that the certain segments log-transformed WSS was independently related to conventional parameters in evaluating diastolic function（mitral lateral E/e', septal E/e', mitral lateral e', septal e' and LAVI）.Conclusions In diastolic period, segmental LV WSS shows a regular variation phenomenon and has specific age- and gender-related patterns in different diastolic phases. The mapping of WSS may help identify the diastolic hemodynamic changes or diastolic function phase by phase.


Introduction
In the past few years, increasing attention has been dedicated to the intraventricular ow pattern. Flow patterns is the consequence of the heart's chiral geometry and the interaction of the lling jet with the walls and mitral valve of the left ventricle (LV), and they reveal the exceptional adaptability of the cardiovascular system for maintaining relatively constant blood circulation under a high workloads [1,2].
The abnormal ow patterns within the ventricular chamber is related to many sorts of LV dysfunction, such as myocardial ischemia [3], cardiomyopathy [4], and thrombosis [5]. Therefore, ow patterns may offer a novel index of LV dysfunction [6].
Vector ow mapping (VFM), which is a combination with the color Doppler and two-dimensional speckle tracking technique, is a novel echocardiographic technology that can visualize the intraventricular ow patterns [7]. Blood ow visualization studies provide clues to reveal physiological and pathophysiological mechanisms by which abnormal ow patterns increase cardiac workload and deteriorate ventricular functions [8,9]. Energy loss (EL), circle, and wall shear stress (WSS), which derived from the intraventricular ow velocity vector eld, are parameters that re ect the spatial dispersion of intraventricular ow patterns. Most previous studies on VFM focused on EL and circle [3,10]. But recently, WSS has been reported to be a new quanti cation parameter [11], because it may indicate the underlying association between uid mechanics and cardiovascular diseases (CVDs)risk [12]. WSS plays a key role in regulating endocardial cells and triggers a series of biological signal transduction, which in turn, regulates gene expression and function of vascular wall cells [13,14]. For example, in the embryonic stage, WSS can in uence the development of the original heart by adjusting the endocardial cells. High blood ow shear force is bene cial to maintain normal vascular endothelial function, and low blood ow shear force often indicates that endothelial function is damaged [15,16].
Although WSS has been reported useful in previous studies, litter was known about its value in evaluating diastolic functions. This study aimed to explore the LV WSS variation in the diastolic period and analyze the differences strati ed by age and gender. Furthermore, to provide a quantitative evaluation of the diastolic LV intracavity blood ow and function.

Study population
A total of 371 healthy volunteers (60.6% women), mean (SD) age was 43 (17), were recruited from the physical examination center of the Second A liated Hospital of Harbin Medical University from August 2018 to March 2019. Afterward, they were separated into four groups according to their age quartiles.
Including criteria were normal at clinical presentation, no history of any comorbidity, and ECG of sinus rhythm.All subjects provided informed consent for study participation and anthracycline therapy administration. This study was approved by the ethics committee ofthe Second A liated Hospital of Harbin Medical University.Con ictso nterest The authors have no con icts of interest to declare.

Image acquisition
The study was performed on Hitachi Aloka LISENDO 880 ultrasound system (Hitachi-Aloka Medical, Ltd, Tokyo, Japan), with the phased array single crystal probe(probe frequency 1.0-5.0 MHz, frame rate 66-78 Hz). All subjects were connected to a 12-lead ECG and examined in the left lateral decubitus position.
Conventional and tissue Doppler transthoracic echocardiography (TTE) was performed by experienced sonographers and reviewed by senior physicians. In two-dimensional (2D) echocardiographic assessment, left ventricular end-diastolic diameter (LVEDd), left ventricular end-systolic diameter (LVESd), end-diastolic septal thickness (IVST), and left ventricular posterior wall thickness (LVPWT) were measured. In PW/TDI evaluation, the early diastolic peak velocity (E) and late diastolic peak velocity (A) of the mitral valve were measured in the apical four-chamber view, with the E/A ratio calculated. The early diastolic peak velocity of mitral annulus (lateral e' and septal e') was also measured for further calculation of lateral E/e' and septal E/e'. The left ventricular ejection fraction (LVEF) was generated by the biplane Simpson method. In terms of VFM mode, the 2D gain was adjusted to optimized visualization of the endocardium, mitral valve, and aortic valve. The size of sampling frame was also adjusted to completely envelop the LV, while the Nyquist limit for CDFI was set high enough so that the ow lled the left ventricle without aliasing and blood ow spilling. The 2D and CDFI images of the apical 4-chamber (Apic 4C), 3-chamber (Apic 3C), and 2-chamber (Apic 2C) views were recorded (Fig. 1).

Data analysis
The acquired images were then imported into the DAS-RS1 workstation for o ine analysis. Firstly, the endocardial border was traced to the clearest frame manually, and then the software automatically traced to the remaining frames. The user was allowed to check and edit the image frame by frame. The diastolic period was de ned as the rst frame after aortic valve closure to the rst frame after mitral valve closure.
And the diastolic period was then divided into four phases based on the ECG, time-ow curve, and twodimensional cardiac valvular opening and closing, including isovolumic diastolic period (D1), rapid lling period (D2), slow lling period (D3), and atrial contraction period (D4).
The WSS images, together with the raw data of the Apic 4C, 3C, and2C, were processed on the o ine VFM workstation, and the raw data were subsequently imported into the WSS segmentation template [see Additional le 1], in which LV was divided into six walls: anterior, anterolateral, inferolateral, inferior, inferoseptal and anteroseptal (Fig. 2). Since the VFM technique is a combination of CDFI and twodimensional speckle tracking, it could acquire both the radial and axial ow velocity, as well as the boundary conditions of bilateral walls, allowing the continuity equation to calculate the intraventricular ow quantitatively ( Figure 3). The Newton inner friction equation of WSS was as follows [17]: WSS μ(Vmax-0) /dy μVmax/dy μ: blood viscosity coe cient 4.0×10 -3 ( N s m -2 ) In the equation, Vmax means the near-wall maximum ow velocity in the cardiac chamber, and the dy means the distance between the point of Vmax and the adjacent ventricular wall. As such, the (Vmax-0)/dy represents the gradient of near-wall ow velocity. And WSS, which is a vector with size and direction, is the product of blood viscosity (μ) and (Vmax-0)/dy [18,19].

Statistical analysis
The SPSS statistic software (IBM SPSS, version 22.0, Chicago, IL) was applied.Continuous data were expressed as mean± standard deviation (SD), and those with skewed distributions are presented as medians (25th and 75th percentiles).Comparisons among different age groups were analyzed using the Kolmogorov-Smirnov test Comparisons between men and women were carried out by the Mann-Whitney tests. Moreover, Log-transformed WSS value in the single LV wall among different phases were compared by one-way repeated measures analysis of variance (ANOVA).The independent correlations between logtransformed WSS and conventional parameters in evaluating diastolic function were explored using stepwise multiple regressions. P <0.05 was considered statistically signi cant.
The study was approved by the Harbin Medical University and was conducted in accordance with the principles of the Declaration of Helsinki

Clinical and conventional echocardiographic parameters
A total of 375 healthy subjects that satis ed the inclusion criteria were recruited originally for this study. However, four were excluded because of the poor image quality, and nally, 371 entered the analysis. The clinical data and echocardiographic measurements of all eligible subjects were listed based on different age groups in Table 1. Peak A velocity of mitral in ow was increased with age, whereas peak E velocity of mitral in ow, lateral e' and septal e' velocity of mitral annulus, E/A, mitral lateral E/e' and septal E/e' decreased with age (P<0.05).  Table 2). Segmental/phase gender differences of WSS could be found. One case Women had greater diastolic WSS than men in anterior and anterolateral segments of phase D2 inferoseptal segments of phase D3 and anterolateral segment of phase D4. and the other casea, men had greater diastolic WSS than women in inferoseptal and anterospetal segment in phase D4. (P<0.05, Table 3).

WSS characteristics in different diastolic phases
For each age group, there were signi cant differences in the log-transformed LV WSS value among different diastolic phases.And the absolute value of the log-transformed WSS appeared the increasingdecreasing--increasing trend from phase D1 to D4, with a peak value at the rapid lling phase (Fig. 4).

Association between WSS and conventional parameters in evaluating diastolic function
Multiple regression analysis was conducted to evaluate the association between diastolic logtransformed LV WSS and the 2D echocardiographic determiners of diastolic function (Tables 4 and 5

Discussion
The temporal and spatial distribution of blood ow velocity contributes to providing the diagnostic and prognostic information of CVDs [20]. The blood ow interferes since the cardiac wall is not smooth and rough. A kind of viscous friction is produced when the near-wall blood ows through the wall due to the viscosity of blood. Thus it is essential to characterize and quantify the ow-wall interaction for evaluating the LV structure and function. WSS is the tangential component of wall friction, which also presents a vector eld that is tangent to the surface anywhere. WSS can quantify the interaction of the intraventricular ow vector and the wall [12], thus re ecting the changes in cardiac structure and function.
Despite the WSS can be measured by MRI [21,22], the clinical application of MRI is limited for its long examination duration, high costs, and low temporal resolution. Echocardiographic VFM technology is a novel visualization approach modi ed based on the method proposed by Garcia et al [18]. It could serve as an ideal tool to visualize the intraventricular ow vector, which may accurately evaluate the local and global hemodynamics during the left ventricular diastolic period, and re ect the corresponding left ventricular diastolic function [23]. In the VFM technology, the Gaussian ltering is employed to remove noise and the non-smooth factors, to smooth the blood ow, without producing lots of errors [18].
The absolute value of WSS in certain segments increased gradually with age, which may be due to the aging-related changes in blood viscosity and ow velocity. Due to the decrease of HR [24] and metabolic requirements [25], the intracardiac ow velocity decreases with the increases of age. However, the prevalence of triglycerides and LDL cholesterol was signi cantly increased with aging [26]. Elevated levels of plasma triglycerides and LDL cholesterol have been found associated with increase in blood viscosity [27]. Our study showed the absolute value change of WSS may be more affected by blood viscosity.There was also a gender discrepancy in WSS values. One plausible reason may be the higher level of testosterone in men, which may shorten the QT interval, and result in the shorter action potential duration [28]. Besides,compared with men, women may have a stronger cardiac response to demand. Thus, gender could present as a factor when evaluating WSS value [29]. All of the above may be the reason why women had greater diastolic WSS than men in most of all related segments in general.The structure and function of LV may change with age and may be affected by different diet, lifestyle, heredity [30]. In our study, WSS is proportional to blood viscosity and intracardiac ow velocity.
The velocity in the LV cavity was lowest compared with other diastolic phases in the period before the mitral valve opening after aortic valve closure. It then increased dramatically with the ow passed from the left atrium into the left ventricle rapidly [3]. The velocity gradually decreased when it turned to the out ow tract after reaching the apex. However, with the left atrium contracting, the ow entered the left ventricle again, resulting in increased ow velocity [3,20,31]. In the present study, the absolute value of LV WSS coincided with the increasing-decreasing-increasing trend during the D1 through D4 phases. This is because the change in WSS was related to blood ow velocity during the diastolic period. According to the WSS calculation formula, WSS also increased with the increase in the velocity changes [19].
According to the 2019 guidelines of the American Society of Echocardiography, E/e',lateral e', septal e' and LAVI were indexes for the assessment of left ventricular diastolic function in normal LVEF subjects [32]. In these four age groups of this study, WSS of some certain LV segments is independently associated with lateral E/e' and septal E/e', lateral e', septal e' and LAVI which indicated that WSS could be a new and helpful parameter to evaluate the left ventricular lling pressure and diastolic function.
It has been showed that the changes in WSS could re ect the changes in vascular and cardiac functions in some diseases [33]. Ji et al. reported the LV vortex and WSS were evaluated in patients with HCM. Their results showed that, compared with the control group, peak values of LV global WSS at rapid ejection phase, atrial contraction phase, and rapid lling phase increased in HCM patients, whereas that value of early diastolic phase decreased in HCM patients [11]. WSS is directly related to the vascular function, and the high WSS could regulate the inner diameter of a blood vessel, and inhibit the increase in blood vascular wall, thrombosis and in ammation. However, a lower WSS is known to express an atherogenic endothelial gene pro le, as observed in the carotid arteries in subjects with risk factors for atherosclerosis [33]. Additionally, WSS also exerts a vital role in the cardiovascular system in hypertension patients, which is achieved by releasing bioactive molecules directly or indirectly [34,35]. Therefore, it is essential to explore the spatial and temporal distribution patterns of WSS during different phases in normal subjects.
Certain limitations should also be noted in this study. Firstly, the VFM technology can only examine the ow eld vector change in the two-dimensional plane, and there is still a certain gap between the accurate measurements of the LV complex three-dimensional ow eld vector change. Secondly, the current study was a preliminary study based on a small study population, while the changes in WSS must be veri ed in a larger and more diverse range. Thirdly, the blood cell contents are different among various individuals, which also results in the different blood viscosity coe cients [7].
To sum up, the LV WSS in normal subjects showed certain changes during the diastolic period in our current cohort, which coincided with the ow changes in LV, and might provide valuable information for the assessment of LV diastolic function. Further study needs to be performed to verify its clinical value in evaluating the diastolic function for both healthy individuals and patients with certain kinds of disease.   The blood ow vector of the left ventricle in the apical view (3a). Vr: the blood ow vector parallel to the sound beam; Vθ: the blood ow vector perpendicular to the sound beam. Correlation between Vr and WSS (3b). The direction of WSS is perpendicular to the wall, from low speed to high speed.

Figure 4
The variation trend of LV WSS at the diastolic period