*2.2. Tissue Preparation*

Frozen left ventricle (LV) tissues were homogenized in CETi lysis buffer (pH 7.6; TransLab, Korea) containing a non-ionic detergent, protease inhibitors (4 mM AEBSF, 1 µg/mL benzamidine, 1 µg/mL leupeptin, 1 µg/mL pepstatin, 1 mM EDTA, and 1 mM EGTA), and phosphatase inhibitors (1 mM sodium fluoride, 1 mM sodium orthovanadate, 1 mM beta-glycerophosphate, and 2.5 mM sodium pyrophosphate). The LV tissues were prepared with the OMNI TH115 Tissue Homogenizer (OMNI International, Kennesaw, GA, USA). The samples were centrifuged at 10,000× *g* twice (20 and 10 min, respectively) at 4 ◦C, and the supernatants were collected for protein analysis.

#### *2.3. Hematoxylin and Eosin Staining*

LV tissues were first subjected to paraffin-embedding; then, the paraffin-embedded tissue blocks were cut into 5-µm-thick cross-sections, and the sections were placed on glass slides. Next, the sections were deparaffinized by xylene treatment and hydrated in running distilled water for at least 2 min. The samples were stained with hematoxylin for 5 min, rinsed with running tap water, and stained with eosin solution for 2 min. The slides were then rinsed with absolute alcohol. They were then mounted using synthetic resin and dried overnight at room temperature. Images of the section were captured on a microscope of Axioplan 2 (Carl Zeiss, Jena, Germany) and the images were quantified using the ImageJ analysis program (NIH, Bethesda, MD, USA). Two sections of LV in 4 rats per group were analyzed for percentage of extramyocyte space, number of myocytes, and cross-sectional area (CSA). Each section was targeted to the endocardial region. The average myocyte count of the sections of the group was calculated per 100,000 µm<sup>2</sup> area, and the mean myocyte CSA for the left ventricular histological section is square micrometers.
