Bmal1 Regulates the Redox Rhythm of HSPB1, and Homooxidized HSPB1 Attenuates the Oxidative Stress Injury of Cardiomyocytes

Figure 5

Bmal1 increased the level of homooxidized HSPB1. (a–f) NRCM isolation and culture methods were mentioned in Methods. (a–c) NRCMs were treated with H₂O₂ at 0, 100, 200, 400, 600, and 800 mM for 120 min, and (d–f) NRCMs were treated with 800 mM of H₂O₂ for 0, 15, 30, 60, 120, and 240 min; (a, d) Bmal1 and β-actin were measured by Western blot analysis, and the homooxidized HSPB1 was analyzed by NEM-alkylated redox Western blot. (b, e) HSPB1 redox potentials were determined using band intensities and the Nernst equation. (c, f) Densitometry was used to determine the fold expression of Bmal1 compared with β-actin. (g–k) H9c2 cells were transfected with 2 μg pCMV-myc-Bmal1 or 15 nM Bmal1-siRNA for 48 h and then treated with different concentrations of H₂O₂ (0 or 800 mM) for 120 min. (g) NEM lysis buffer was used for extraction of total protein. (h) HSPB1 redox potentials were determined using band intensities and the Nernst equation. Relative mRNA expression of Glcm (i), Txnrd1 (j), and HMOX1 (k) in H9c2 cells compared with the control. Graphs are representatives of three independent experiments. ; , , and .

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