Protective role of triiodothyronine in sepsis‑induced cardiomyopathy through phospholamban downregulation

Figures

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  Figure 1. Serum T3 levels are significantly negatively correlated with myocardial PLN levels in sepsis. (A) Hematoxylin and eosin staining of mouse myocardial tissues. Yellow arrows indicate inflammatory cells. Scale bar, 10 µm; n=6. (B) Masson staining was applied to mouse myocardial tissues. Scale bar, 10 µm; n=6. ELISA of the serum levels of (C) cTnI and (D) T3 (n=6). WB of PLN protein levels and semi-quantitative analysis in the (E) myocardial tissues of mice (n=6) and in (F) H9C2 cells (n=3). Quantitative PCR analysis of the mRNA expression levels of PLN in the (G) myocardial tissues of mice (n=6) and in (H) H9C2 cells (n=3). (I) ELISA measured the serum levels of PLN in the serum of mice (n=6). (J) Spearman correlation analysis of T3 and PLN concentrations in mouse serum. All experiments were repeated three times. Data are presented as the mean ± standard deviation. *P<0.05, **P<0.01, ****P<0.0001. cTnI, cardiac troponin I; LPS, lipopolysaccharide; PLN, phospholamban. qPCR, quantitative PCR; T3, triiodothyronine.

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  Figure 2. T3 treatment alleviates cardiomyocyte damage in vitro. (A) Cell Counting Kit-8 assay assessed H9C2 cell viability (n=3). (B) Apoptosis detection and statistical analysis of H9C2 cells (n=3). (C) Crystal violet staining and statistical analysis of H9C2 cells (n=3). (D) Quantitative PCR detected the mRNA expression levels of IL-6 and TNF-α in H9C2 cells (n=3). (E) Fluo-4AM fluorescence images of H9C2 intracellular calcium levels. Scale bar, 50 µm; n=3. (F) DHE fluorescence images of H9C2 intracellular reactive oxygen species levels. Scale bar, 10 µm; n=3. (G) Flow cytometric detection of (G) Fluo-4 AM and (H) DHE mean fluorescence intensity in H9C2 cells (n=3). All experiments were repeated three times. Data are presented as the mean ± standard deviation. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. IL-6, interleukin 6; LPS, lipopolysaccharide; OD, optical density; PI, propidium iodide; PLN, phospholamban; T3, triiodothyronine; TNF-α, tumor necrosis factor α.

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  Figure 3. T3 treatment alleviates cardiomyocyte damage in vivo. (A) Serum ELISA results for mice (n=6). (B) Quantitative PCR detection of mRNA expression levels in mice (n=6). (C) H&E staining was applied to mouse myocardial tissue (n=6). Yellow arrows indicate inflammatory cells. Scale bar, 10 µm. (D) Masson staining and quantitative analysis were applied to mouse myocardial tissue (n=6). Scale bar, 10 µm. All experiments were conducted in triplicate using independent samples. The data are presented as the mean ± standard deviation. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. cTnI, cardiac troponin I; H&E, hematoxylin and eosin; LPS, lipopolysaccharide; PLN, phospholamban; SERCA2, sarcoplasmic reticulum calcium ATPase; T3, triiodothyronine.

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  Figure 4. T3 reduces PLN expression through JNK/c-Jun signaling pathway inhibition. WB detection of protein levels in the (A) myocardial tissues of mice (n=6) and in (B) H9C2 cells (n=3). (C) Quantitative PCR detection of mRNA expression levels in H9C2 cells (n=3). (D) WB detection of protein levels in H9C2 cells (n=3). All in vitro experiments were performed using triplicate samples and were repeated three times. Data are presented as the mean ± standard deviation. **P<0.01, ***P<0.001, ****P<0.0001. LPS, lipopolysaccharide; NC, negative control; OE, overexpression; p-, phosphorylated; PLN, phospholamban; SERCA2, sarcoplasmic reticulum calcium ATPase; T3, triiodothyronine; WB, western blotting.

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  Figure 5. Inhibition of the JNK/c-Jun signaling pathway improves cardiomyocyte damage. (A) Western blotting detection of protein levels and semi-quantitative analysis in H9C2 cells (n=3). (B) Apoptosis assay and statistical analysis in H9C2 cells (n=3). (C) Quantitative PCR detection of mRNA expression levels in H9C2 cells (n=3). (D) Fluorescence images of H9C2 intracellular DHE reactive oxygen species levels. Scale bar, 10 µm; n=3. (E) Fluo-4 AM fluorescence images of H9C2 intracellular calcium levels. Scale bar, 50 µm; n=3. All experiments were repeated three times. Data are presented as the mean ± standard deviation. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. IL-6, interleukin 6; LPS, lipopolysaccharide; p-, phosphorylated; PI, propidium iodide; PLN, phospholamban; SP, SP600125; TNF-α, tumor necrosis factor α.

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  Figure 6. Clinical value of elevated PLN in patients with SIC. (A) Levels of T3 and PLN in each group of patients were measured by ELISA. (B) Receiver operating characteristic curves were constructed according to the serum levels of PLN, cTnI and BNP. (C) Correlation curves of serum PLN with PCT, ALB and CREA. Data are presented as the mean ± standard deviation. ns, no significance; *P<0.05, **P<0.01, ****P<0.0001. ALB, albumin; AUC, area under the curve; BNP, brain natriuretic peptide; CREA, creatinine; cTnI, cardiac troponin I; PCT, procalcitonin; PLN, phospholamban; SIC, sepsis-induced cardiomyopathy; T3, triiodothyronine