The Overexpression of miR‐377 Aggravates Sepsis‐Induced Myocardial Hypertrophy by Binding to Rcan2 and Mediating CaN Activity

Figures

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  Figure 1. MiR-377 is highly expressed in myocardial tissues of mice with CLP-induced sepsis. (a) Evaluation of cardiomyocyte hypertrophy in CLP-induced septic mice by echocardiography and ventricular body mass ratio (n = 9), ∗p < 0.05 compared with sham-operated mice. (b) HE staining of myocardial tissues of mice in sham-operated mice and CLP-induced septic mice group. (c) Myocardial tissue morphology of mice in sham-operated mice and CLP-induced septic mice, as observed by transmission electron microscope. (d) The expression level of miR-377 in the myocardial tissues in sham-operated mice and CLP-induced septic mice, as measured by qRT-PCR. ∗p < 0.05 compared with sham-operated mice. (e) The level of inflammatory factors and serum myocardial enzyme indexes of myocardial tissues in sham-operated mice and CLP-induced septic mice, as examined by ELISA. ∗p < 0.05 compared with sham-operated mice. Measurement data were showed as mean ± standard deviation. Data of two-group were analyzed by unpaired t-test.

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  Figure 2. MiR-377 repression could ameliorate cardiomyocyte hypertrophy in septic mice induced by CLP. (a) LVPW, LVESD, EF, and FS detected by echocardiography and HW and LVW/BW measured by weighing, reflecting the successful establishment of a sepsis mouse model, wherein the sepsis was induced by CLP. (b) Myocardial damage of mice in each group, as detected by HE staining. (c) Myocardial tissue morphology of mice in each group, as observed by transmission electron microscope. (d) The expression of inflammatory factors in myocardial tissues of mice, as evaluated by ELISA assay. ∗p < 0.05 compared with sham-operated mice. #p < 0.05 compared with the antagomir NC group. &p < 0.05 compared with the antagomir NC group. Measurement data were represented as mean ± standard deviation. One-way ANOVA with Tukey's posthoc test was employed to analyze data of multiple groups. n = 9, mice in each group.

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  Figure 3. MiR-377 negatively regulated Rcan2 level (n = 9). (a) The sequence of 3′-UTR region of miR-377 and Rcan2 gene. (b) The results of double luciferase reporter gene experiment. (c) Heat map analyzed by GSE9667 microarray dataset. (d) The level of miR-377 and Rcan2 in hypertrophic myocardial tissue of mice, as measured by qRT-PCR. (e) The expression of Rcan2 in hypertrophic myocardial tissue of mice, as detected by Western blot. ∗p < 0.05 compared with the mimic NC group. #p < 0.05 compared with the inhibitor NC group. Measurement data were expressed as mean ± standard deviation. The data comparison between multiple groups was performed by ANOVA. Unpaired t-test was adopted to analyze the data between two groups. Above experiments were repeated in triplicate.

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  Figure 4. MiR-377 modulated CaN activity by restricting Rcan2 (n = 9). (a) The mRNA level of Rcan2 and CaN in hypertrophic myocardial tissues of mice, as examined by qRT-PCR. (b) The protein expression of Rcan2 and CaN in hypertrophic myocardial tissues of mice, as measured by Western blot. ∗p < 0.05 compared with the oe-NC group. #p < 0.05 compared with the oe-Rcan2 group. &p < 0.05 compared with the si-NC group. @p < 0.05 compared with the si-Rcan2 group. Measurement data were represented as mean ± standard deviation. Comparisons between multiple groups were performed by one-way ANOVA, followed by Tukey's posthoc test. Above experiments were repeated in triplicate. n = 9, mice in each group.

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  Figure 5. MiR-377 increased CLP-induced cardiomyocyte hypertrophy in septic mice by inhibiting Rcan2 and regulating CaN activity through Ca2+-CaN pathway. (a) [Ca2+]i in cardiomyocytes of each group. (b) The level of miR-377, Rcan2, and CaN, as measured by qRT-PCR. (c) The protein expression of Rcan2 and CaN, as examined by Western blot. (d) The result of cell proliferation in each group as examined by EdU assay. (e) The result of cell apoptosis in each group as examined by flow cytometry. (f) The expression level of inflammatory factors (TNF-α, IL-6, and IL-8) quantified by ELISA assay. ∗p < 0.05 compared with the mimic NC group. #p < 0.05 compared with the miR-377 mimic group. &p < 0.05 compared with the inhibitor NC group. @p < 0.05 compared with the miR-377 inhibitor group. Measurement data were showed as mean ± standard deviation. Data of multiple groups were compared by one-way ANOVA with Tukey's posthoc test. All experiments were repeated for 3 times.