Seven-week-old male Dahl salt-sensitive rats (Japan SLC, Shizuoka, Japan) were housed under conditions of constant temperature (22℃) and humidity (60%), exposed to a 12-hour light/dark cycle, and offered tap water to drink. Rats were divided into two groups: hypertension group (n = 6), which were fed a high-salt (8% NaCl) diet for 9 weeks to develop LV hypertrophy, and control group (n = 6), which were fed a normal diet. All surgery was performed under anesthesia, and all efforts were made to minimize suffering. All protocols for animal experiments were approved in accordance with the recommendations of the Okayama University Animal Care and Use Committee, and all methods were performed in accordance with the relevant guidelines and regulations. This study was reported in accordance with the ARRIVE guidelines.
Transthoracic echocardiography was performed using Aplio ver. 6.0 with a 10-MHz sector probe (Canon Medical Systems, Otawara, Japan). The rats were anesthetized from inhalation of 2% isoflurane while lying in a left recumbent position. LV end-diastolic and end-systolic diameters were measured in the short-axis view, and fractional shortening was calculated. Interventricular septum and LV free wall thicknesses were measured.
Shear wave elasticity.
SW imaging was performed by ex vivo experiment. A retrograde perfusion system was used to maintain the rat’s heart in a completely relaxed state 14. After sacrifice under inhalation of 2% isoflurane anesthesia, the heart was quickly excised and submerged in the Tyrode solution (136 mmol/L NaCl, 5.4 mmol/L KCl, 1.8 mmol/L CaCl2, 0.53 mmol/L MgC12, 5.5 mmol/L HEPES, and 1% Glucose, pH 7.4, 37℃) with 20 mmol/L butanedione monoxime, an inhibitor of actin-myosin interaction, and 10 µmol/L blebbistatin, a specific myosin II inhibitor. The ascending aorta was cannulated with an 18-gauge blunted needle connected to a retrograde perfusion system. The heart was perfused with the Tyrode solution with butanedione monoxime and blebbistatin to induce complete relaxation. The heart was set in a water tank of agar phantom.
SW elasticity was measured using Aplio i900 with an 18-MHz linear probe (Canon Medical Systems). B-mode image was obtained in the long-axis view. A rectangular region of interest was placed on LV free wall. SW was generated by pushing pulse, and SW velocity was obtained based on the tissue Doppler technique (Fig. 1). After confirming a proper SW propagation in “wave front” style display, a circular region of interest of 1-mm in diameter was placed on the image. SW elasticity was measured automatically using the equation: 3ρc2 (c: SW velocity, ρ: tissue density). Each measurement was repeated five times, and the average value was calculated.
The heart was sectioned transversely at the mid-papillary level, and then fixed with 10% formalin, embedded in paraffin, and cut into 5-µm-thick sections. Sections were stained with hematoxylin-eosin for assessing myocardial hypertrophy, and with picrosirius red for assessing myocardial fibrosis. The cross-sectional area of cardiomyocytes was quantitatively measured at 50 locations using ImageJ software (version 1.52v, National Institutes of Health, Bethesda, MD, USA), and the average value was calculated. The extent of myocardial fibrosis was also measured, and the percentage was calculated.
Data are presented as mean ± standard deviation for continuous variables. Variables were compared by unpaired t-test. Relationships of SW elasticity with myocardial hypertrophy and fibrosis were analyzed by Pearson’s correlation coefficient. Statistical analysis was performed with JMP version 14.2 (SAS Institute Inc., Cary, NC, USA), and significance was defined as a value of P < 0.05.