Four-month-old sex-matched WT and β2a-Tg mice were given either normal chow (WT-N and β2a-N) with normal water or a high-fat diet (HFD) with/without l -NAME in water (WT-HFD, WT-LN, WT-HFD-LN, β2a-HFD, β2a-LN, and β2a-HFD-LN) for 4 mo. SAHA treatment was given to another group of 4-mo-old sex-matched β2a-Tg mice together with HFD and l -NAME (β2a-HFD-LN-SAHA) for 4 mo as well. Echocardiography was performed at baseline (4 mo old, before treatment) and once a month after different treatments. Long-term animal survival and body weight (BW) were recorded during the 4-mo follow-up (Supplemental Fig. S1).
After 4 mo of treatment, mice were terminated using inhaled isoflurane (Butler Shein Animal Health, Dublin Ohio) after measurement of hemodynamics parameters. BW weight was recorded at terminal time points. Organs, including heart, lung, liver, kidney, and spleen, were carefully trimmed and collected, rinsed with PBS, and weighed after blotting off the excess fluid. Organ weight-to-body weight ratios were calculated. Heart tissues were excised and cut into two parts (basal and apical). The basal part containing part of papillary muscles was fixed with 10% formalin, then paraffin embedded for histology following previously described protocols (23 (link), 24 (link)). The apical part was snap-frozen in liquid nitrogen for molecular analysis. Plasma samples were collected for further study. The analysis of histology such as cardiomyocyte cross-sectional area (CSA), Picrosirius red staining, and echocardiography was performed by investigators blinded to groups.
After 4 mo of treatment, mice were terminated using inhaled isoflurane (Butler Shein Animal Health, Dublin Ohio) after measurement of hemodynamics parameters. BW weight was recorded at terminal time points. Organs, including heart, lung, liver, kidney, and spleen, were carefully trimmed and collected, rinsed with PBS, and weighed after blotting off the excess fluid. Organ weight-to-body weight ratios were calculated. Heart tissues were excised and cut into two parts (basal and apical). The basal part containing part of papillary muscles was fixed with 10% formalin, then paraffin embedded for histology following previously described protocols (23 (link), 24 (link)). The apical part was snap-frozen in liquid nitrogen for molecular analysis. Plasma samples were collected for further study. The analysis of histology such as cardiomyocyte cross-sectional area (CSA), Picrosirius red staining, and echocardiography was performed by investigators blinded to groups.
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