The Antioxidant Selenoprotein T Mimetic, PSELT, Induces Preconditioning-like Myocardial Protection by Relieving Endoplasmic-Reticulum Stress

Abstract

Oxidative stress and endoplasmic reticulum stress (ERS) are strictly involved in myocardial ischemia/reperfusion (MI/R). Selenoprotein T (SELENOT), a vital thioredoxin-like selenoprotein, is crucial for ER homeostasis and cardiomyocyte differentiation and protection, likely acting as a redox-sensing protein during MI/R. Here, we designed a small peptide (PSELT), encompassing the redox site of SELENOT, and investigated whether its pre-conditioning cardioprotective effect resulted from modulating ERS during I/R. The Langendorff rat heart model was employed for hemodynamic analysis, while mechanistic studies were performed in perfused hearts and H9c2 cardiomyoblasts. PSELT improved the post-ischemic contractile recovery, reducing infarct size and LDH release with and without the ERS inducer tunicamycin (TM). Mechanistically, I/R and TM upregulated SELENOT expression, which was further enhanced by PSELT. PSELT also prevented the expression of the ERS markers CHOP and ATF6, reduced cardiac lipid peroxidation and protein oxidation, and increased SOD and catalase activities. An inert PSELT (I-PSELT) lacking selenocysteine was ineffective. In H9c2 cells, H₂O₂ decreased cell viability and SELENOT expression, while PSELT rescued protein levels protecting against cell death. In SELENOT-deficient H9c2 cells, H₂O₂ exacerbated cell death, that was partially mitigated by PSELT. Microscopy analysis revealed that a fluorescent form of PSELT was internalized into cardiomyocytes with a perinuclear distribution. Conclusions: The cell-permeable PSELT is able to induce pharmacological preconditioning cardioprotection by mitigating ERS and oxidative stress, and by regulating endogenous SELENOT.

Keywords: antioxidants; heart; ischemia/reperfusion injury; peptides; selenoproteins.

Figure 1

Effect of the selenoprotein T-derived peptide 43-52 (PSELT) and tunicamycin (TM), alone and in combination, on systolic and diastolic functions of Langendorff-perfused hearts subjected to I/R injury. (A) dLVP and (B) LVEDP variations. Black boxes indicate ischemic administration (Bonferroni multiple comparison test). dLVP = 6.97 % of total variation between groups (p < 0.001); LVEDP = 20.70 % of total variation between groups (p < 0.001). dLVP and LVEDP values at the end of reperfusion are showed in the inset graph. Data are expressed as changes of dLVP and LVEDP values (millimeters of mercury) from stabilization to the end of the 120 min of reperfusion with respect to the baseline values for control (I/R alone), PSELT, TM, TM + PSELT (n = 6 hearts/group) and I-PSELT groups (n = 4 hearts). p < 0.01 (**), p < 0.001 (***) (one-way ANOVA and Newman–Keuls multiple comparison test).

Figure 2

Effect of PSELT and TM, alone and in combination, on infarct size and LDH activity in coronary effluent of Langendorff-perfused hearts subjected to I/R injury. (A) Infarct size (IS) for I/R, PSELT, TM, TM + PSELT (n = 6 hearts/group) and I-PSELT groups (n = 4 hearts). The amount of necrotic tissue measured after 30 min global ischemia and 120 min reperfusion is expressed as a percentage of the LV mass (%IS/LV). p < 0.05 (*), p < 0.01 (**), p < 0.001 (***) (one-way ANOVA and Newman–Keuls multiple comparison test). (B) LDH activity was determined in coronary effluent 5 min before ischemia and 10, 20 and 30 min after ischemia in the reperfusion phase. LDH variations for I/R, PSELT, TM, TM + PSELT (n = 6/group) and I-PSELT groups (n = 4) (Bonferroni multiple comparison test). A total of 3.26 % of total variation between groups (p < 0.001). Inset graph shows LDH activity at the end of 10, 20 and 30 min of reperfusion. Data are expressed as IU/mL for I/R, PSELT, TM, TM + PSELT (n = 6/group) and I-PSELT groups (n = 4). p < 0.05 (*), p < 0.01 (**), p < 0.001 (***) (one-way ANOVA and Newman–Keuls multiple comparison test).

Figure 3

Effect of PSELT and TM, alone and in combination, on lipid peroxidation, protein oxidation, and antioxidant enzymes. (A) TBARS levels, (B) protein carbonyl groups, (C) SOD enzyme activity and (D) CAT enzyme activity in sham and I/R-exposed hearts for I/R alone, PSELT, TM, TM + PSELT (n = 6/group) and I-PSELT groups (n = 4). p < 0.05 (*), p < 0.01 (**), p < 0.001 (***) (one-way ANOVA and Newman–Keuls multiple comparison test).

Figure 4

Effect of PSELT and TM, alone and in combination on protein expression of SELENOT and ERS-related markers. Western blot analysis of (A) SELENOT, (B) CHOP, and (C) ATF6 in sham and I/R-exposed hearts for I/R alone, PSELT, TM, TM + PSELT and I-PSELT groups (n = 4 hearts/group). Histograms represent the ratio of densitometric analysis of protein/loading control. p < 0.05 (*), p < 0.01 (**), p < 0.001 (***) (one-way ANOVA and Newman–Keuls multiple comparison test).

Figure 5

Effects of PSELT against oxidative stress-induced damage in H9c2 cells. (A) Effect of PSELT on cell viability in H9c2 cells exposed to a pre-treatment with increasing concentration of PSELT (5, 10, 25, 50, 100, 500, and 1000 nM) or (B) inert PSELT (I-PSELT) (5, 10, 25, 50, 100, 500, and 1000 nM) for 24 h and then exposed to H₂O₂ (200 μM) for additional 3 h. Cell viability was assessed using MTT assay. Results are represented as mean ± SEM (n = 6 per group). Significant differences were detected by one-way ANOVA followed by Newman–Keuls multiple comparison test, p < 0.05 (*), p < 0.01 (**), p < 0.001 (***). (C) Western blot analysis of SELENOT in H9c2 cells exposed to vehicle (indicated as control) or pre-treated with increasing concentration of PSELT (5, 10, 25, 50, 100, 500, and 1000 nM) for 24 h and then exposed to H₂O₂ (n = 3 independent experiments) (rabbit polyclonal antibody against SELENOT and β-actin. Histograms represent the ratio of densitometric analysis of protein:loading control. Significant differences were detected by t-test. p < 0.05 (*), p < 0.01 (**). (D) Representative image of SELENOT knockdown in H9c2 cells, transfected with 100 nM siRNA-SELENOT or negative control (siRNA-NC). Data are shown as mean ± SEM from three independent experiments. Histograms represent the ratio of densitometric analysis of protein:loading control. Significant differences were detected by t-test. p < 0.05 (*) vs. si-NC group. (E) Effect of si-SELENOT gene silencing on cell viability in H9c2 cells pre-treated with increasing concentration of PSELT for 24 h and exposed to H₂O₂ for 3 h. Cell viability was evaluated using MTT assay and was expressed as the percentage of control cells only transfected with siRNA negative control (indicated as si-NC). Results are represented as mean ± SEM (n = 6 per group). Significant differences were detected by one-way ANOVA followed by Newman–Keuls multiple comparison test. p < 0.001 (***).

Figure 6

Evaluation of PSELT ability to penetrate into the cardiomyocyte and its intracellular distribution. Representative confocal microscopic images of H9c2 cardiomyocytes incubated for 15 min with (A) vehicle or (B) PSELT-dansyl (blue) 30 nM. Laminin antibody (Sigma Aldrich) was used as plasma membrane marker (green) and propidium iodide was used for nuclei staining (red). Scale bars: 12.5 μm. (C) Quantification of relative immunofluorescence of PSELT-dansyl in control and treated groups. Data are expressed as mean ± SEM [unpaired t-test, **** p < 0.001 (n = 4 per group)].

Figure 7

Schematic representation of the mechanism of PSELT-induced preconditioning-like cardioprotection. The ability of cell-penetrating PSELT to protect the heart against I/R insult, in the presence and absence of TM-induced functional ERS activation is illustrated. PSELT preconditioned the heart by (i) inhibiting lipid peroxidation and protein oxidation; (ii) enhancing SOD and CAT activities; (iii) relieving TM-induced ERS markers CHOP and ATF6. Dashed line indicates putative mechanisms, in particular related to the potential cooperative role of endogenous SELENOT in the PSELT-dependent cardioprotection. For further explanation, see text. ATF6: activating transcription factor 6; CAT: catalase; CHOP: C/EBP homologous protein; ER: endoplasmic reticulum; ERS: endoplasmic reticulum stress; I/R: ischemia/reperfusion; ROS: reactive oxygen species; SELENOT: selenoprotein T; SOD: superoxide dismutase; TM: tunicamycin.