Histologic Analysis

Mouse hearts were harvested and then fixed in 4% paraformaldehyde for 48 hours. Fixed hearts were embedded in paraffin and cut into 5-μm sections. Heart sections were stained with hematoxylin and eosin or wheat germ agglutinin to evaluate hypertrophic growth. Hematoxylin and eosin staining was performed using a standard method. For wheat germ agglutinin staining, deparaffinized and rehydrated heart sections were boiled in 10 mmol/L citrate buffer (pH 6.0) for 12 minutes, followed by blocking in 1% bovine serum albumin with 5% goat serum for 1 hour. Sections were then incubated with 10 μg/mL Alexa Fluor 594-conjugated wheat germ agglutinin ({"type":"entrez-nucleotide","attrs":{"text":"W11262","term_id":"1285567","term_text":"W11262"}}W11262; Thermo Fisher) for 1 hour at room temperature in the dark. After mounting with the ProLong gold antifade reagent ({"type":"entrez-protein","attrs":{"text":"P36935","term_id":"549826","term_text":"P36935"}}P36935; Thermo Fisher), sections were imaged with a fluorescence microscope (Leica). Circular to oval left ventricular myocytes were chosen from >5 mice. At least 10 fields per mouse were evaluated. Cell size was quantified using Image-Pro Plus software.