MicroRNAs Involved in the Mitogen-Activated Protein Kinase Cascades Pathway During Glucose-Induced Cardiomyocyte Hypertrophy  E. Shen, Xuehong Diao, Xiaoxia.

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MicroRNAs Involved in the Mitogen-Activated Protein Kinase Cascades Pathway During Glucose-Induced Cardiomyocyte Hypertrophy  E. Shen, Xuehong Diao, Xiaoxia Wang, Ruizhen Chen, Bing Hu  The American Journal of Pathology  Volume 179, Issue 2, Pages 639-650 (August 2011) DOI: 10.1016/j.ajpath.2011.04.034 Copyright © 2011 American Society for Investigative Pathology Terms and Conditions

Figure 1 Glucose causes cardiac hypertrophy of diabetic mice and cardiomyocytes hypertrophy of neonatal rat. A: Heart weight to body weight ratios (HW/BW) from diabetic mice (n = 15) and nondiabetic mice (n = 10). B: Morphological analysis of hearts from diabetic mice and nondiabetic mice. Scale bar = 1 mm (top). Hematoxylin-eosin–stained micrographs showing transverse sections of left ventricular myocardium. Original magnification, ×40. Scale bar = 25 μm (bottom) C: Measurement of cardiomyocyte cross-sectional area sampled from heart sections (n = 5 for each group). D: mRNA expression of ANP, BNP, and β-MHC in cardiac hypertrophy induced by STZ (n = 5). E: Micrograph images of cultured rat neonatal myocytes treated with 25 mmol/L glucose (HG), 25 mmol/L L-glucose (osmotic), or 5 mmol/L glucose (NG) after 48 hours (left panels). Cell surface areas of more than 100 individualized cells per condition were measured using ImageJ software (right panel). *P < 0.05 compared with control or NG. The American Journal of Pathology 2011 179, 639-650DOI: (10.1016/j.ajpath.2011.04.034) Copyright © 2011 American Society for Investigative Pathology Terms and Conditions

Figure 2 Pathway analysis based on miRNA-targeted genes, which showed significant pathways targeted by aberrantly expressed miRNAs. The vertical axis is the pathway category; the horizontal axis is the enrichment of pathways. The American Journal of Pathology 2011 179, 639-650DOI: (10.1016/j.ajpath.2011.04.034) Copyright © 2011 American Society for Investigative Pathology Terms and Conditions

Figure 3 MicroRNA–gene network. Square represents miRNAs; circle represents target genes; straight line represents miRNA–gene relationship. The size of the square represents the degrees of miRNAs; larger degrees have larger numbers of target genes. The American Journal of Pathology 2011 179, 639-650DOI: (10.1016/j.ajpath.2011.04.034) Copyright © 2011 American Society for Investigative Pathology Terms and Conditions

Figure 4 The interaction between MAPK signaling pathway–related genes and miRNAs. Square represents miRNAs; circle represents target genes; straight line represents miRNAs-MAPK signaling pathway–related gene relationship. The American Journal of Pathology 2011 179, 639-650DOI: (10.1016/j.ajpath.2011.04.034) Copyright © 2011 American Society for Investigative Pathology Terms and Conditions

Figure 5 Activation of JNK, ERK1/2, and p38 MAPK in cardiomyocyte hypertrophy induced by hyperglycemia. A: Western blot analysis of JNK, ERK1/2, and p38 MAPK protein levels in cardiac tissue of diabetic mice (upper panels). Results are shown for 3 mice in each group. B: Western blot analysis of ERK1/2, JNK, and P38 MAPK in neonatal rat cardiomyocytes (upper panels). Data are mean ± SD from at least three different cell cultures. For A and B, bar graph (lower panels) shows expression ratio of phospho-JNK over total JNK, phospho-ERK over total ERK, and phospho-p38 over total p38, respectively. *P < 0.05 compared with control or NG. The American Journal of Pathology 2011 179, 639-650DOI: (10.1016/j.ajpath.2011.04.034) Copyright © 2011 American Society for Investigative Pathology Terms and Conditions

Figure 6 Neonatal rat cardiomyocytes were exposed to high glucose and transfected with miR-373 mimic. A: miR-373 expression levels in cardiomyocytes 48 hours posttransfection were determined using real-time PCR. miRNA mimic negative controls were used for control transfections. *P < 0.05 compared with control-transfected cardiomyocytes. B: Overexpression of miR-373 decreased cell surface area as compared with HG. C: Western blot analysis of MEF2C in miR-373–overexpressing cardiomyocytes (upper panels). Quantitative analysis MEF2C protein levels by densitometry (lower panels). *P < 0.05 compared with HG; **P < 0.05 compared with NG. The American Journal of Pathology 2011 179, 639-650DOI: (10.1016/j.ajpath.2011.04.034) Copyright © 2011 American Society for Investigative Pathology Terms and Conditions

Figure 7 Effect of miR-373 mimic on expression of JNK, ERK, and p38 MAPK. Representative Western blot of phospho-JNK and total JNK, phospho-ERK1/2 and total ERK1/2, and phospho-p38 and total p38 in myocytes after miR-373 mimic transfected (upper panels, A–C). For A–C, bar graph (lower panels) shows expression ratio of phospho-JNK over total JNK, phospho-ERK1/2 over total ERK1/2, and phospho-p38 over total p38, respectively. *P < 0.05 compared with control or NG. The American Journal of Pathology 2011 179, 639-650DOI: (10.1016/j.ajpath.2011.04.034) Copyright © 2011 American Society for Investigative Pathology Terms and Conditions

Figure 8 The effect of p38 inhibitor on cardiac myocyte hypertrophy, miR-373, and MEF2C expression. A: Cardiac myocyte size treated with or without p38 inhibitor in the presence or absence of high glucose. B: Quantitative analysis of cardiomyocyte surface area. C: Effect of p38 inhibitor on miR-373 expression levels in cardiomyocytes. D: Representative Western blot of MEF2C protein expression levels (upper panels). Quantitative analysis MEF2C protein levels by densitometry (lower panels). *P < 0.05 compared with HG; **P < 0.05 compared with NG. The American Journal of Pathology 2011 179, 639-650DOI: (10.1016/j.ajpath.2011.04.034) Copyright © 2011 American Society for Investigative Pathology Terms and Conditions