The redox-active defensive Selenoprotein T as a novel stress sensor protein playing a key role in the pathophysiology of heart failure

Abstract

Maladaptive cardiac hypertrophy contributes to the development of heart failure (HF). The oxidoreductase Selenoprotein T (SELENOT) emerged as a key regulator during rat cardiogenesis and acute cardiac protection. However, its action in chronic settings of cardiac dysfunction is not understood. Here, we investigated the role of SELENOT in the pathophysiology of HF: (i) by designing a small peptide (PSELT), recapitulating SELENOT activity via the redox site, and assessed its beneficial action in a preclinical model of HF [aged spontaneously hypertensive heart failure (SHHF) rats] and against isoproterenol (ISO)-induced hypertrophy in rat ventricular H9c2 and adult human AC16 cardiomyocytes; (ii) by evaluating the SELENOT intra-cardiomyocyte production and secretion under hypertrophied stimulation. Results showed that PSELT attenuated systemic inflammation, lipopolysaccharide (LPS)-induced macrophage M1 polarization, myocardial injury, and the severe ultrastructural alterations, while counteracting key mediators of cardiac fibrosis, aging, and DNA damage and restoring desmin downregulation and SELENOT upregulation in the failing hearts. In the hemodynamic assessment, PSELT improved the contractile impairment at baseline and following ischemia/reperfusion injury, and reduced infarct size in normal and failing hearts. At cellular level, PSELT counteracted ISO-mediated hypertrophy and ultrastructural alterations through its redox motif, while mitigating ISO-triggered SELENOT intracellular production and secretion, a phenomenon that presumably reflects the extent of cell damage. Altogether, these results indicate that SELENOT could represent a novel sensor of hypertrophied cardiomyocytes and a potential PSELT-based new therapeutic approach in myocardial hypertrophy and HF.

Keywords: Antioxidants; Cardiac dysfunction; Cardiomyocytes; Peptides; Selenoproteins.

Fig. 1

Effects of PSELT on systemic inflammation and cardiac dysfunction. Plasma levels of A IL-1β and B TNF-α. C Flow cytometry analysis of M1 polarization marker CD80 in RAW 264.7 macrophages untreated (unstained cells, UC) or treated with vehicle (CTRL), LPS (100 ng/ml), LPS + PSELT and PSELT (5 nM) for 24 h. Histograms represent the percentages of CD80 positive cells (n = 4 independent experiments). Plasma levels of D LDH and E BNP, and cardiac levels of F Gal-3 (n = 6) in WST, WST + PSELT, SHHF, SHHF + PSELT groups. Data are expressed as means ± SEM. Significant differences were detected by one-way ANOVA and Newman-Keuls multiple comparison test. *p < 0.05; **p < 0.01; ***p < 0.001

Fig. 2

Effect of PSELT on cardiac ultrastructure and on protein expression of SELENOT and fibrosis/senescence-related markers in cardiac tissues. A Ultrastructure representations obtained by transmission electron microscopy (TEM) on cardiac sections of control animals (WST) and SHHF animals chronically treated with or without PSELT (scale bar: 2 μm). B Western blot analysis of desmin in soluble and insoluble fractions of WST, WST + PSELT, SHHF, SHHF + PSELT hearts. C Gelatin SDS-PAGE zymographic analysis of MMP-2 activity in myocardial tissues. Western blot analysis of CTGF, p53, p21, γH2AX, SELENOT, in the hearts of WST, WST + PSELT, SHHF, SHHF + PSELT groups (n = 3). Histograms represent the ratio of densitometric analysis of protein:loading control. Data are expressed as the mean ± SEM (n = 3 independent experiments). Significant differences were detected by one-way ANOVA and Newman-Keuls multiple comparison test. *p < 0.05; **p < 0.01, ***p < 0.001

Fig. 3

Effect of PSELT on cardiac function and infarct size (IS) of Langendorff-perfused rat hearts subjected to I/R injury. A dLVP and B LVEDP variations. Gray boxes indicate ischemic administration (Bonferroni multiple comparison test). dLVP = 21.98% of total variation between groups (p < 0.001); LVEDP = 19.46% of total variation between groups (p < 0.001). Inset histograms show dLVP and LVEDP at the end of reperfusion. Data are expressed as changes of dLVP and LVEDP values (millimeters of mercury, mmHg) from stabilization to the end of reperfusion with respect to the baseline values for WST, WST + PSELT, SHHF, SHHF + PSELT groups (n = 6 hearts/group). Significant differences were detected by one-way ANOVA and Newman-Keuls multiple comparison test. **p < 0.01, ***p < 0.001. C Infarct size (IS) expressed as a percentage of the LV mass (% IS/LV) in WST, WST + PSELT, SHHF, SHHF + PSELT groups. Significant differences were detected by one-way ANOVA and Newman-Keuls multiple comparison test. **p < 0.01, ***p < 0.001

Fig. 4

Effects of PSELT on cardiomyocytes hypertrophy induced by isoproterenol in vitro. A Morphological staining of H9c2 cells exposed to vehicle (saline, NaCl 0.9%) indicated as a CTRL, isoproterenol (ISO) (100 µM), ISO + PSELT and PSELT (5 nM) for 48 h and relative quantification of cell surface area performed by ImageJ. Scale bars: 25 μm. Data are expressed as mean ± SEM. Significant differences were detected by one-way ANOVA and Newman-Keuls multiple comparison test. ***p < 0.001. Evaluation of mRNA expression levels of B NppA (ANP) and C NppB (BNP), by qPCR in H9c2 cardiomyocytes exposed to vehicle (CTRL), ISO, ISO + PSELT and PSELT for 48 h. Samples were analyzed in duplicate (n = 3 independent experiments). The relative mRNA expression levels of the hypertrophic genes were normalized to 18S rRNA. Fold change is calculated on the basis of the 2^−ΔΔCT. Data are expressed as the mean ± SEM. Significant differences were detected by one-way ANOVA and Newman-Keuls multiple comparison test. *p < 0.05; **p < 0.01, ***p < 0.001. D Morphological staining of H9c2 cells exposed to vehicle (saline, NaCl 0.9%) indicated as a CTRL, isoproterenol (ISO) (100 µM), ISO + I-PSELT and I-PSELT (5 nM) for 48 h and relative quantification of cell size performed by ImageJ. Scale bars: 25 μm. Data are expressed as mean ± SEM and significant differences were detected by one-way ANOVA and Newman-Keuls multiple comparison test. **p < 0.01; ***p < 0.001

Fig. 5

Effects of ISO and PSELT on SELENOT expression and role of endogenous protein on cell hypertrophy in H9c2 cardiomyocytes. A Western blot analysis of SELENOT expression in H9c2 cells exposed to vehicle (saline, NaCl 0.9%) indicated as a CTRL, isoproterenol (ISO) (100 µM), ISO + PSELT and PSELT (5 nM). Histograms represent the ratio of densitometric analysis of protein:loading control. Data are expressed as the mean ± SEM (n = 3 independent experiments). Significant differences were detected by one-way ANOVA and Newman-Keuls multiple comparison test. *p < 0.05. B Morphological staining of H9c2 cells transfected with Negative control si-RNA (NC si-RNA) or Selenoprotein T si-RNA (SELENOT si-RNA) for 36 h and then treated with or without ISO (100 µM) for additional 48 h. Scale bars: 25 μm. Cell surface area (%) was quantified by ImageJ. Data are expressed as mean ± SEM from three independent experiments. Significant differences were detected by one-way ANOVA and Newman-Keuls multiple comparison test. **p < 0.01; ***p < 0.001. C Representative Western blot analysis of SELENOT in H9c2 cardiomyocytes transfected with NC si-RNA or SELENOT si-RNA for 36 h. Histograms represent the ratio of densitometric analysis of protein:loading control. Data are expressed as the mean ± SEM (n = 3 independent experiments). Significant differences were detected by t-test. *p < 0.05

Fig. 6

Effects of PSELT against cell hypertrophy induced by isoproterenol in vitro. A F-actinin morphological staining of AC16 human cardiomyocytes exposed to vehicle (saline, NaCl 0.9%) indicated as a CTRL, isoproterenol (ISO) (100 µM), ISO + PSELT and PSELT (5 nM) for 48 h and relative quantification of cell surface area performed by ImageJ. Scale bars: (a, b, c, d) 25 μm and (a1, b1, c1, d1) 12.5 μm. Data are expressed as mean ± SEM. Significant differences were detected by one-way ANOVA and Newman-Keuls multiple comparison test. **p < 0.01. Evaluation of mRNA expression levels of B NPPA (ANP) and C NPPB (BNP), by qPCR in AC16 human cardiomyocytes exposed to vehicle (CTRL), ISO, ISO + PSELT and PSELT for 48 h. Samples were analyzed in duplicate (n = 3 independent experiments). The relative mRNA expression levels of the hypertrophic genes were normalized to 18S rRNA. Fold change is calculated on the basis of the 2^−ΔΔCT. Data are expressed as the mean ± SEM. Significant differences were detected by one-way ANOVA and Newman-Keuls multiple comparison test. *p < 0.05; **p < 0.01. D F-actinin staining of AC16 cells exposed to vehicle (saline, NaCl 0.9%) indicated as a CTRL, isoproterenol (ISO) (100 µM), ISO + I-PSELT and I-PSELT (5 nM) for 48 h and relative quantification of cell size performed by ImageJ. Scale bars: (a, b, c, d) 25 μm and (a1, b1, c1, d1) 12.5 μm. Data are expressed as mean ± SEM and significant differences were detected by one-way ANOVA and Newman-Keuls multiple comparison test. ***p < 0.001

Fig. 7

Effects of isoproterenol (ISO) and PSELT on Selenoprotein T (SELENOT) intracellular and extracellular levels in human cardiomyocytes. A Intra-cardiomyocyte levels of SELENOT in AC16 cells exposed to vehicle (saline, NaCl 0.9%) indicated as a CTRL, isoproterenol (ISO) (100 µM), ISO + PSELT and PSELT (5 nM) for 24–48-72–96 h. B SELENOT levels in cell culture medium (supernatant) of AC16 cardiomyocytes exposed to vehicle (saline, NaCl 0.9%) indicated as a CTRL, isoproterenol (ISO) (100 µM), ISO + PSELT and PSELT (5 nM) for 24–48-72–96 h. Data are expressed as means ± SEM. Significant differences were detected by one-way ANOVA and Newman-Keuls multiple comparison test. *p < 0.05; **p < 0.01; ***p < 0.001

Fig. 8

Ultrastructure representations obtained by transmission electron microscopy (TEM) on AC16 human cardiomyocytes exposed to ISO and PSELT, alone or in combination. A Control group (scale bar: 2 μm, magnification × 4000); B ISO group (scale bar: 2 μm, magnification × 4000) and B₁ (scale bar: 1 μm, magnification × 8000; C ISO + PSELT group (scale bar: 2 μm, magnification × 4000) and C₁ (scale bar: 1 μm, magnification × 8000; D PSELT group (scale bar: 2 μm, magnification × 4000); E quantitative TEM analysis showing the number of Golgi stacks per cell that was determined by a counting grid method from 10 different electron microscope images for each sample; F quantitative TEM analysis of mitochondrial cristae number based on the quantification of the cristae per mitochondrial section from different mitochondria. Only mitochondria with intact and completely visible borders have been included in the analysis