WGA Staining

We performed standard wheat germ agglutinin (WGA) staining on 5 μm cryosections of the heart from the four groups of mice: WT, Akita, Akita treated with miR-133a mimic (Akita+miR), and Akita treated with scrambled miRNA (Akita+scm). The details of WGA staining are elaborated in our previous publication. In brief, we prepared heart cryosections by using CryoStar NX50 (Thermo Fisher Scientific, Waltham, MA, United States) and fixed them in 4% paraformaldehyde. We washed them with phosphate buffered saline (PBS) and then incubated them with 5 μg/ml of WGA (cat # W834, Thermo Fisher Scientific, Waltham, MA, United States) for 10 min at room temperature. They were then washed, mounted with coverslip, and observed under a fluorescence microscope (EVOS, Life Technologies, United States). Representative WGA stainings of the heart from the four groups are presented in Figures ​Figures3A,3A, ​,7A7A. We measured mean cardiomyocyte diameter (μm) in high magnification (40×) image. We randomly selected five areas (within the black open circle in Figures ​Figures3C,3C, ​,7C7C) in the left ventricle and scored total 50 cells/per mouse from the five areas (10 cells/area). The mean cardiomyocytes diameter was quantified to determine cardiomyocytes hypertrophy.

Measurement of cardiac hypertrophy in Akita. (A) Representative Wheat Germ Agglutinin (WGA) staining of the heart sections. Green color represents cardiomyocyte boundary and “^∗” denotes a typical cardiomyocyte size. (B) Quantification of cardiomyocytes diameter. Values are presented as mean ± SD. N = 3 per group. Total number of cells scored for hypertrophy was 50. A minimum of five different fields of left ventricle was used from each mice. Two-tailed, unpaired Student’s t-test was used for statistical analysis. P < 0.05 is considered statistically significant. (C) Representative hematoxylin–eosin staining of left ventricular section. Outlined circles represent the area used for hypertrophic analysis.

miR-133a mimic treatment mitigates cardiac hypertrophy in Akita. (A) Representative Wheat Germ Agglutinin (WGA) staining of the heart (left ventricle) sections. Green color represents cardiomyocyte boundary and “^∗” denotes a typical cardiomyocyte size. (B) Quantification of cardiomyocytes diameter. Values are presented as mean ± SD. N = 3 per group. Total number of cells scored for hypertrophy was 50. A minimum of five different fields of left ventricle was used from each mice. Two-tailed, unpaired Student’s t-test was used for statistical analysis. P < 0.05 is considered statistically significant. (C) Representative hematoxylin–eosin staining of left ventricular section. Outlined circles represent the area used for hypertrophic analysis.