1 INTRODUCTION

Hypertension is one of the major risk factors affecting the morbidity and mortality of cardiovascular diseases. Long-term hypertension has been established to directly induce and aggravate the irreversible deterioration of left ventricular (LV) function.1, 2 At present, the application of treadmill exercise stress echocardiography (TESE) in subclinical cardiac function and related reserve evaluation of essential hypertension still need to be improved, most reports just focus on global myocardium rather than different layers.3, 4 A number of studies have shown that when the LV ejection fraction (EF) is within the normal range, speckle-tracking strain analysis can be used for the early detection of subclinical myocardial function injury, and represents superior predictor of adverse outcome.5-7 As an emerging indicator, the layer-specific strain (LSS) has attracted much attention in clinical application and has been achieved to quantify.8 Rowin and colleagues showed that TESE had higher sensitivity and specificity in the diagnosis of suspected myocardial functional damage proposing this approach as an important new technique for noninvasive evaluation.9 Therefore, TESE was applied to evaluate the left ventricular LSS in subclinical myocardial function and peak reserve characteristics of patients with essential hypertension.

2 METHODS 2.1 Study population and inclusion criteria

A total of 55 patients (average age 54 ± 7.2 years) with high blood pressure constituted the hypertensive patient group, the inclusion criteria: According to the European Guidelines for the Management of Hypertension10 with blood pressure ≥140/90 mmHg or who explicitly took antihypertensive drugs. Patients were excluded: there was definitive evidence of coronary artery disease (stenosis ≥50%), stent implantation, diabetes, congenital heart disease, cardiomyopathy of any kind, cardiac valvular disease, arrhythmia, or poor imaging quality. A second sex and age-matched group (51 ± 9.4 years) consisting of 51 healthy person with normal range blood pressure (<140/90 mmHg) were used as controls. Patients who participated in TESE were discontinued from β-blockers for 48 h and caffeine or theophylline intake for 12 h. The study was approved by ethics review committee of the hospital , and all patients signed informed consent before examination.

2.2 TESE and image acquisition

A General Electrics (GE) Medical System E95 ultrasound system was employed to acquire the study data using the adult heart M5s probe (frequency 1.5–5 MHz). All participants received a complete transthoracic echocardiography examination at rest and peak status. Five standards (4-, 2-, and 3-chamber, papillary muscle level of short axis, parasternal long axis) views were obtained. TESE was performed using the standard Bruce protocol and the metabolic equivalent (MET)11 was used to derive the metabolic tolerance index. A 12-lead ECG was used to monitor subjects throughout the process with blood pressure recorded every 2 min. The target exercise heart rate was 85% of 220-age, peak blood pressure rises of more than 210 mmHg, obvious electrocardiogram changes (ST-segment horizontal or oblique depression more than .1 mV), and intolerable chest pain or fatigue were indications to terminate the test. Immediately after exercise, subjects were instructed to lie in the lateral decubitus position and peak images collect for about 2–3 min. Image collection and data analysis were completed by the same physician.

2.3 Speckle tracking image analysis

Analyses were carried out according to the methods recommended by the American Society of Echocardiography (ASE) guidelines. The images were analyzed offline after transferring to the GE-Echo PAC workstation. Calculated parameters included the LV end-diastolic volume index (LVEDVI); LV end diastolic diameter (LVDd); interventricular septum diastolic diameter (IVSd), LV posterior wall diastolic diameter (LVPWd); LV ejection fraction (LVEF); s,E/A,e/a and E/e values from transmitral flow spectrum and mitral annular motion curve were measured at rest and peak state for the two groups and LV mass index (LVMI) was calculated according to the Devereux formula.12

GE-Q-analysis technology was used to manually track and correct the endocardial margin. The area of interest in each segment covered the entire thickness of the myocardial wall and avoided inclusion of the pericardium. The software automatically generated the outline curve to obtain layer strain data of speckle-tracking in resting and peak stress, respectively. The longitudinal layer-specific strain (LLSS) and circumferential layer-specific strain (CLSS) were divided into three layers: endocardium (Endo), mid-myocardium (Mid), and epicardium (Epi). The characteristics of LLSS and CLSS in three apical (4-, 2-, and 3-chamber) views and parasternal short-axis view at the papillary muscle level were compared between two groups in resting and peak status, respectively. The parameters of GLS and CS myocardial deformation were evaluated at three layer-specific levels. The reserve function of strain change from rest to peak was evaluated with absolute contractile reserve (CR) calculated as the difference in multi-layer strain between the peak and their corresponding resting values.

2.4 Statistical analysis

Conventional ultrasonic parameters with continuous variables including left ventricular LSS and absolute CR values were shown as the mean ± SD. The data of two groups were compared using independent sample t-test. The Chi-square test was used for comparisons of case numbers and gender. ANOVA with Bonferroni correction was used to compare the values of strain between each layer of myocardium. Pearson correlation coefficient was used to analyze relationships between two parameters. The intraclass correlation coefficient (ICC) of 10 patients were randomly selected for consistency evaluation in the same observer and different observers. An ICC greater than .75 indicated good consistency. The Endo, Mid, and Epi layer strain differences between the same and different observers were 10.9%, 11.4%, 12.9%, and 10.7%, 13.5%, 14.6%, respectively. SPSS25.0 software was used for all analyses and p < .05 was considered to indicate statistically significant differences.

3 RESULTS 3.1 Study population and general echocardiographic data

Comparisons of the demographic and basic clinical characteristics of the hypertensive and control subject groups established there were no significant differences in gender, mean age, body surface area (BSA) and body mass index (BMI). As anticipated, systolic BP for rest and peak measurements were higher in the hypertensive group along with lower exercise METs. The detailedly use of antihypertensive medications, prevalence of hypercholesterolaemia, and duration of hypertension were showed (Table 1).

TABLE 1. Demographic and clinical characteristics of the study population Control group (N = 51) Hypertension group (N = 55) t/χ2 value p-Value Age (years) 51.2 ± 9.4 54.0 ± 7.2 −4.451 .761 Male (%) 58.8 52.7 .398 .528 BSA (m2) 1.6 ± .25 1.7 ± .28 −1.537 .136 BMI 21.2 ± 4.5 23 ± 3.9 −3.454 .234 Duration of hypertension (≥5 years) (%) – 26 (47.3) – – Medications (%) – – – Aspirin 17 (30.9) β-blockers 16 (29.1) Calcium channel blockers 19 (34.5) ACEI 6 (10.9) ARB 9 (16.3) Statin 18 (32.7) Hypercholesterolemia (%) 3 (5.9) 9 (16.4) 2.896 .089 SBP-rest (mmHg) 113.3 ± 17.2 136.7 ± 12.6 −4.641 <.001 SBP-peak (mmHg) 135.8 ± 14.6 157.9 ± 16.7 −7.774 <.001 METs 8.5 ± 2.1 7.0 ± 1.6 2.142 .022 Abbreviations: ACEI, angiotensin-converting enzyme inhibitor; ARB, angiotensin receptor blocker; BMI, body mass index; BSA, body surface area; MET, metabolic equivalent; SBP, systolic blood pressure.

We next compared the general ultrasound parameters for resting and peak states are summarized in Table 2. There were significant differences observed in LVDd, IVSd, PWd and LVMI, as well as LVEDVI-rest between the two groups. Both s-peak and HR-peak values were significantly lower in the hypertensive group, while no differences occurred in LVEF. For the hypertensive group, E/e ratios were significantly higher versus control subjects, which suggests that the diastolic reserve function was reduced, particularly evident by the increased E/e ratios at peak exercise.

TABLE 2. Echocardiographic characteristics of the study population Control group (N = 51) Hypertension group (N = 55) t-Value p-Value LVMI (g/m2) 98.5 ± 9.9 129.4 ± 11.3 −9.083 <.001 LVDd (mm) 44.7 ± 2.8 47.9 ± 3.7 −2.219 .027 IVSd (mm) 9.8 ± .5 12.1 ± .8 −4.823 .013 PWd (mm) 9.4 ± .6 11.5 ± 1.0 −6.547 <.001 LVEDVI-rest 43.1 ± 6.9 47.5 ± 8.5 2.411 .022 (m3/m2)-peak 39.7 ± 8.4 41.3 ± 8.9 3.537 .079 E/e-rest 6.5 ± .67 8.5 ± .36 −4.352 <.001 E/e-peak 5.8 ± .78 12.1 ± .38 −5.891 <.001 EF-rest .65 ± .08 .63 ± .04 1.896 .527 EF-peak .82 ± .07 .80 ± .05 .511 .382 HR(bpm)-rest 69.2 ± 16.3 65.3 ± 19.2 1.889 .069 HR(bpm)-peak 125.4 ± 16.5 120.5 ± 21.6 5.347 .021 s (cm/s)–rest .14 ± .03 .12 ± .04 1.024 .214 s (cm/s)-peak .20 ± .04 .16 ± .03 4.621 .045 Abbreviations: HR, heart rate; IVSd, interventricular septum diameter; LVDd, left ventricular end diastolic diameter; LVEDVI, Left ventricle end-diastolic volume index; LVEF, left ventricular ejection fraction; LVMI, left ventricular mass index; PWd, posterior wall diameter; E,early diastolic mitral flow (pulsed Doppler); e,average of the peak early diastolic relaxation velocity of the septal and lateral mitral annulus (tissue Doppler); s,average of the peak systolic velocity of the septal and lateral mitral annulus (tissue Doppler). Data are presented as mean ± SD. 3.2 LSS difference characteristics between hypertensive and normal group

Tables 3 and 4 depicts the difference in the three LSS values at four/two/three and the papillary muscle level chamber views. Both LLSS and CLSS values showed a gradually decreasing trend from the endocardial to epicardial layers, and revealed different degrees of reduction in hypertensive group, especially the endocardial strain which was significantly decreased both at rest and peak states. At rest, comparisons for 4LSendo, 4LSmid, 3LSendo, 3LSmid, 2LSendo were significantly lower in hypertensive group (p < .01), whereas each LLSS values were lower (p < .01) except 3LSepi at peak exercise exercise. Example for the resting GLSendo was (control group 24.4% ± 1.5% vs. hypertensive group 20.4% ± 2.3%), while the difference was more obvious at peak exercise (control vs. hypertensive group, 30.8% ± 2.8% and 22.8% ± 2.9%, respectively).

TABLE 3. Comparison of longitudinal LSS in different views Longitudinal strain (%) Normal group Hypertensive group t-Value p-Value 4Endo rest 24.0 ± 2.7 20.9 ± 3.2 6.543 <.001 peak 30.3 ± 3.3 22.8 ± 3.0 9.427 <.001 4Mid rest 20.6 ± 1.4 17.1 ± 1.8 6.833 <.001 peak 26.1 ± 3.2 20.4 ± 3.0 10.165 <.001 4Epi rest 18.6 ± 1.4 16.3 ± 2.9 1.923 .116 peak 22.7 ± 3.0 18.9 ± 2.9 7.117 <.001 2Endo rest 24.3 ± 1.5 21.0 ± 2.5 4.779 <.001 peak 32.2 ± 2.6 23.5 ± 3.4 13.671 <.001 2Mid rest 21.1 ± 1.4 18.8 ± 2.2 1.290 .605 peak 28.1 ± 2.3 21.2 ± 3.0 11.725 <.001 2Epi rest 18.4 ± 1.5 17.7 ± 2.0 1.138 .982 peak 25.6 ± 2.3 20.5 ± 2.5 7.792 <.001 3Endo rest 24.2 ± 1.5 20.1 ± 2.6 7.454 <.001 peak 30.1 ± 2.5 22.0 ± 2.4 6.973 <.001 3Mid rest 20.8 ± 1.1 17.8 ± 2.6 4.388 <.001 peak 25 ± 2.2 20.9 ± 2.5 6.549 <.001 3Epi rest 18.5 ± 1.0 16.6 ± 2.7 .178 .077 peak 21.4 ± 2.2 18.5 ± 2.2 2.328 .054 GLS-Endo rest 24.4 ± 1.5 20.4 ± 2.3 7.346 <.001 peak 30.8 ± 2.8 23.3 ± 2.9 10.177 <.001 GLS-Mid rest 20.9 ± 1.3 17.8 ± 2.1 6.125 <.001 peak 26.7 ± 2.5 19.8 ± 2.8 8.162 <.001 GLS-Epi rest 18.4 ± 1.6 16.8 ± 2.4 1.750 .349 peak 23.2 ± 2.5 18.3 ± 2.7 3.866 <.001 p-Value -rest <.001 .007

-peak

(GLS between layers)

<.001 .042 Abbreviations: The endo/mid/epi layer strain value at the four chamber view (4endo, 4mid, 4epi); the endo/mid/epi layer strain value at the two chamber view (2endo, 2mid, 2epi); the endo/mid/epi layer strain value at the three chamber view (3endo, 3mid, 3epi). GLS, global longitudinal strain; GLS-endo, the average value of GLS in the endocardium layer at the four, two, and three chamber views. GLS-mid, the average value of GLS in the midcardium layer at the three different views; GLS-epi, the average value of GLS in the epicardium layer at the three different views; LSS, layer-specific strain. Data are presented as mean ± SD. TABLE 4. Comparison of circumferential LSS Circumferential strain (%) Normal group Hypertensive group t-Value p-Value CSEndo rest 36.3 ± 3.3 31.3 ± 2.6 15.445 <.001 peak 39.2 ± 2.5 33.1 ± 2.7 12.478 <.001 CSMid rest 27.3 ± 2.6 23.4 ± 2.7 8.545 <.001 peak 31.3 ± 2.6 24.2 ± 2.6 9.087 <.001 CSEpi rest 16.2 ± 2.4 14.7 ± 2.4 2.066 .272 peak 20.2 ± 2.7 17.1 ± 3.1 6.648 <.001 p-Value -rest <.001 <.001

-peak

(CS between layers)

<.001 <.001 Abbreviations: CSendo/CSmid/CSepi, the endo/mid/epi circumferential strain value at the papillary muscle level; LSS, layer-specific strain. Data are presented as mean ± SD.

There were also significant differences in CSendo and CSmid between the two groups at rest, while more pronounced differences occured during peak exercise in the three circumferential layers. Subsequently, the parameters of GLS and CS myocardial deformation were evaluated at three layer-specific levels (Tables 3 and 4), revealed lower GLS endocardial-to-epicardial gradient both at rest and peak in hypertensive group (p < .05). Furthermore, comparisons of LSS contractile reserve values between the two groups showed significant differences (p < .01). Absolute CR value was calculated as the difference in multi-layer strain between the peak and their corresponding resting values. The absolute increased multi-layer strain value in hypertensive group was lower, suggesting that CR function of the three layers were all impaired (Table 5).

TABLE 5. Comparison of LS and CS contractile reserve value Contractile reserve value Normal group Hypertensive group t-Value p-Value LSendo-CR 6.4 ± .21 2.9 ± .27 7.645 <.001 LSmid-CR 5.8 ± .22 2 ± .20 8.468 <.001 LSepi-CR 4.8 ± .19 1.5 ± .19 9.039 <.001 GLS-CR 5.3 ± .21 2.1 ± .27 6.742 <.001 CSendo-CR 2.9 ± .29 1.8 ± .23 5.947 <.001 CSmid-CR 4.0 ± .27 .8 ± .18 7.491 <.001 CSepi-CR 4.2 ± .32 2.4 ± .22 2.783 <.001 Abbreviations: CR, contractile reserve; CR was calculated as the difference in multi-layer strain between the peak and their corresponding resting values. LSendo/LSmid/LSepi, the average longitudinal strain value of the endocardium/midcardium/epicardium layer. GLS, global longitudinal strain. CSendo/CSmid/CSepi, the average circumferential strain value of the endocardium/midcardium/epicardium layer at the papillary muscle level. Data are presented as mean ± SD. 3.3 LSS difference characteristics between rest and peak excercise

We evaluated the detailed strain characteristics between resting and peak state conditions. While there were significant increases in each LLSS and CLSS in normal group at peak states (p < .05), among the hypertensive group, only 4LSmid, 4LSepi, 2LSepi, 3LSmid, 3LSepi, and CSepi showed significant increase (p < .05) between resting and peak states (Figures 1 and 2). Variation characteristics of LSS from rest to peak also indicating the CR function of hypertensive group was reduced.