MiR-133a Mimic Alleviates T1DM-Induced Systolic Dysfunction in Akita: An MRI-Based Study

Figures

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  FIGURE 1. Schematic showing treatment regimen of Akita. (A) Lenti-miR-133a was created by packaging miR-133a mimic with lentivirus protein in 293FT cells and they were injected into Akita mice at the age of 12 weeks. (B) Lenti-scrambled was created in the same manner as Lenti-miR-133a except for replacing miR-133a mimic with scrambled miRNA. They were treated to Akita in the same manner as Lenti-miR-133a. (C) Flow schematic showing the timeline for genotyping, mice treatment, performance of cardiac-MRI, and sacrifice. WT, N = 5; Akita, N = 5; Akita+miR-133a, N = 5; and Akita+scm, N = 5.

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  FIGURE 2. Measurement of cardiac dysfunction in Akita by MRI. (A) 2D cardiac performance analysis-MR tracking of left ventricle using long-axis MRI. The global longitudinal strains were recorded. (B) Analyses of cardiac ejection fraction by long-axis MRI. Values are represented as mean ± SD, N = 4–5 per group. (C) Representatives of short-axis images of the heart from WT and Akita mice. Two-tailed, unpaired Student’s t-test was used for statistical analysis. P < 0.05 is considered statistically significant.

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  FIGURE 3. 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.

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  FIGURE 4. Measurement of cardiac fibrosis in Akita by picrosirius red and Masson’s Trichrome stainings. (A) Representative images of picrosirius red stained heart sections (left ventricle region) of WT and Akita mice. (B) Representative images of Masson’s Trichrome stained heart sections of WT and Akita mice. Arrow shows collagen deposition (blue) in the heart. PV, perivascular area and INT, interstitial area (C,D). Quantification of percentage fibrosis (pixel blue color area/total pixel area) by Image J in the four groups. Values are presented as mean ± SD. N = 3 per group was randomly selected for the fibrosis analyses. Two-tailed, unpaired Student’s t-test was used for statistical analysis. P < 0.05 is considered statistically significant.

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  FIGURE 5. Relative miR-133a expression by the individual miR-133a assay. RT-qPCR for miR-133a was performed on the left ventricle tissue of the heart using U6 snRNA as endogenous control. Akita+miR represents Akita treated with Lenti-miR-133a, and Akita+scm represents Akita treated with Lenti-scrambled miRNA. Values are represented as mean ± SEM, N = 3 per group. One-way ANOVA was used for statistical analysis followed by Tukey’s multiple comparisons test. P < 0.05 was considered as statistically significant.

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  FIGURE 6. miR-133a mimic treatment improves systolic dysfunction of Akita. (A) 2D cardiac performance analysis-MR tracking of left ventricle using long-axis MRI. The global longitudinal strains were recorded. (B) Analyses of cardiac percentage ejection fraction by long-axis MRI. (C) Representatives of short-axis and long-axis images of the heart. Values are represented as mean ± SD, N = 4–5 per group. Two-tailed, unpaired Student’s t-test was used for statistical analysis. P < 0.05 is considered statistically significant.

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  FIGURE 7. 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.

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  FIGURE 8. miR-133a mimic treatment alleviates cardiac fibrosis in Akita (A). Representative images of picrosirius red stained heart sections (left ventricle region) of Akita treated with miR-133a mimic (Akita+miR) or scrambled miRNA (Akita+scm). (B) Representative images of Masson’s Trichrome staining of the heart sections of Akita+miR and Akita+scm. Arrow shows the collagen deposition (blue) in the heart. PV, perivascular area; INT, interstitial area (C,D). Quantification of percentage fibrosis (pixel blue color area/total pixel area) by Image J in the four groups. Values are presented as mean ± SD. N = 3 per group was randomly selected for the fibrosis analyses. Two-tailed, unpaired Student’s t-test was used for statistical analysis. P < 0.05 is considered statistically significant.

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  FIGURE 9. Schematics showing that T1DM decreases the cardiac levels of miR-133a, which induces cardiac hypertrophy and fibrosis and decreases percentage ejection fraction (% EF) that ultimately results in cardiac dysfunction and heart failure. MiR-133a mimic treatment mitigates T1DM-induced cardiac hypertrophy and fibrosis, increases EF and ameliorates cardiac dysfunction/heart failure.