Cardiac Nuclear High-Mobility Group Box 1 Ameliorates Pathological Cardiac Hypertrophy by Inhibiting DNA Damage Response

Figures

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  Figure 1. Nuclear HMGB1 and p-ATM Expression in Failing Human Heart (A to C) Representative images and analysis of immunostaining of high-mobility group box 1 (HMGB1) (green), phosphorylation of ataxia telangiectasia mutated (p-ATM) (red), and DAPI (blue) in normal human hearts and failing human hearts. The number of HMGB1- and p-ATM–positive cardiomyocytes (CMs) per field was counted. Scale bars = 20 μm. Results are presented as the mean ± SE. (n = 5 per group). ∗p < 0.05; ∗∗∗p < 0.001. (D to F) Negative correlation between nuclear HMGB1-positive CM and cardiomyocyte diameter (MyD), collagen volume fraction (CVF), or serum brain natriuretic peptide (BNP) levels (n = 32).

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  Figure 2. Cardiac Nuclear HMGB1 Protects Mice Against Ang II–Induced Cardiac Remodeling (A, B) Representative images and analysis of CM cross-sectional area from hematoxylin and eosin (H&E)–stained left ventricular sections in wild-type (WT) and HMGB1 transgenic (HMGB1-Tg) mice following saline or angiotensin II (Ang II) infusion. Scale bars = 20 μm. (n = 6 per group). (C) The ratio of heart weight to tibial length (HW/TL) in WT and HMGB1-Tg mice following saline or Ang II infusion (n = 10 per group). (D, E) Representative images and analysis of cardiac fibrosis area by Masson’s trichrome staining in left ventricular sections in WT and HMGB1-Tg mice following saline or Ang II infusion. Scale bars = 50 μm. n = 5 in WT saline group; n = 4 in WT Ang II group; n = 5 in HMGB1-Tg saline group; n = 5 in HMGB1-Tg Ang II group. Results are presented as the mean ± SE. ∗∗p < 0.01; ∗∗∗p < 0.001. Abbreviations as in Figure 1.

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  Figure 3. Cardiac Nuclear HMGB1 Prevents Ang II–Induced DNA Damage and the DDR in Mice (A, B) Representative images and analysis of immunostaining of γ-H2AX (green), α-actinin (red), and DAPI (blue) in saline- or Ang II–treated WT and HMGB1-Tg mice. The number of γ-H2AX–positive CMs per field was counted. Scale bars = 50 μm. n = 6 in WT saline group; n = 6 in WT Ang II group; n = 6 in HMGB1-Tg saline group; n = 4 in HMGB1-Tg Ang II group. (C, D) Representative images and analysis of Western blots of γ-H2AX and β-tubulin from hearts of WT and HMGB1-Tg mice following saline or Ang II infusion (n = 5 per group). (E, F) Representative images and analysis of immunostaining of p-ATM (red), α-actinin (green), and DAPI (blue) in saline- or Ang II–treated WT and HMGB1-Tg mice. The number of p-ATM–positive CMs per field was counted. Scale bars = 50 μm. n = 6 in WT saline group; n = 7 in WT Ang II group; n = 5 in HMGB1-Tg saline group; n = 6 in HMGB1-Tg Ang II group. (G, H) Representative images and analysis of Western blots of p-ATM and total ATM from hearts of WT and HMGB1-Tg mice following saline or Ang II infusion (n = 6 per group). Results are presented as the mean ± SE. ∗p < 0.05; ∗∗p < 0.01;…

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  Figure 4. Effect of HMGB1 Overexpression on DNA Damage and DDR After Ang II Stimulation (A, B) Representative images and analysis of immunostaining of nuclear foci of γ-H2AX (green), α-actinin (red), and DAPI (blue) of neonatal rat CMs (NRCMs) transfected with pcDNA-HMGB1 or control vector and then treated with saline or Ang II for 24 h. The foci number of γ-H2AX per NRCM was counted. Scale bars = 20 μm. n = 6 per group. (C, D) Representative images and analysis of Western blots of γ-H2AX. NRCMs transfected with pcDNA-HMGB1 or control saline and then treated with vehicle or Ang II for 24 h (n = 5 per group). (E, F) Representative images and analysis of immunostaining of nuclear foci of p-ATM (red), α-actinin (green), and DAPI (blue) of NRCMs transfected with pcDNA-HMGB1 or control vector and then treated with saline or Ang II for 24 h. The foci number of p-ATM per NRCM was counted. Scale bars = 20 μm. n = 5 in control vector saline group; n = 5 in control vector Ang II group; n = 4 in pcDNA-HMGB1 saline group; n = 5 in pcDNA-HMGB1 Ang II group. (G, H) Representative images and analysis of Western blots of p-ATM and total ATM. NRCMs transfected with pcDNA-HMGB1 or control vector and then…

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  Figure 5. Effect of HMGB1 Suppression on DNA Damage and DDR After Ang II Stimulation (A, B) Representative images and analysis of immunostaining of nuclear foci of γ-H2AX (green), α-actinin (red), and DAPI (blue) of NRCMs transfected with small interfering HMGB1 (siHMGB1) or nonspecific control small interfering ribonucleic acid (siRNA) and then treated with saline or Ang II for 24 h. The foci number of γ-H2AX per NRCM was counted. Scale bars = 20 μm. n = 6 in scramble saline group; n = 6 in scramble Ang II group; n = 5 in siHMGB1 saline group; n = 6 in siHMGB1 Ang II group. (C, D) Representative images and analysis of Western blots of γ-H2AX. NRCMs transfected with siHMGB1 or nonspecific control siRNA and then treated with saline or Ang II for 24 h (n = 6 per group). (E, F) Representative images and analysis of immunostaining of nuclear foci of p-ATM (red), α-actinin (green), and DAPI (blue) of NRCMs transfected with siHMGB1 or nonspecific control siRNA and then treated with saline or Ang II for 24 h. The foci number of p-ATM per NRCM was counted. Scale bars = 20 μm. n = 5 in scramble saline group; n = 4 in scramble Ang II group; n = 5 in siHMGB1 saline group; n = 4 in siHMGB1 Ang II…

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  Figure 6. Effect of HMGB1 and ATM on Cardiac Hypertrophic Signaling (A, B) Representative images and analysis Western blots of phospho-extracellular signal-related kinase 1/2 (p-ERK1/2), total ERK1/2 (t-ERK1/2), phospho-nuclear factor kappa B (p-NF-κB), and total NF-κB (t-NF-κB) from NRCMs transfected with pcDNA-HMGB1 or control vector and then treated with saline or Ang II for 4 h (n = 6 per group). (C, D) Representative images and analysis of Western blots of p-ERK1/2, t-ERK1/2, p-NF-κB, and t-NF-κB from NRCMs transfected with siHMGB1 or nonspecific control siRNA and then treated with saline or Ang II for 4 h (n = 6 per group). (E) Representative Western blots of p-ERK1/2, t-ERK1/2, p-NF-κB, and t-NF-κB. NRCMs transfected with siHMGB1 or nonspecific control siRNA were pretreated with an ATM inhibitor (KU55933) or dimethyl sulfoxide (DMSO) for 1 h before Ang II stimulation, and then stimulated with saline or Ang II for 4 h. (F, G) Quantification of atrial natriuretic peptide (ANP) and BNP promoter activities. Rat H9C2 cells were pretreated with an ATM inhibitor (KU55933) or DMSO for 1 h before Ang II stimulation, and then stimulated with saline or Ang II for 24 h (n = 6 per group).…

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  Figure 7. Cardioprotective Effect of the ATM Inhibitor on Ang II–Induced Pathological Cardiac Remodeling (A) Timeline of Ang II infusion and ATM inhibitor (KU55933) or vehicle administration in mice. (B) The ratio of HW/TL in WT and HMGB1-Tg mice subjected to Ang II infusion and vehicle or ATM inhibitor (KU55933) treatment (n = 8 in WT vehicle group; n = 8 in WT Ang II + vehicle group; n = 6 in HMGB1-Tg Ang II + vehicle group; n = 6 in WT Ang II + KU55933 group; n = 6 in HMGB1-Tg Ang II + KU55933 group). (C) Analysis of CM cross-sectional area by H&E staining in left ventricular sections in WT and HMGB1-Tg mice subjected to Ang II infusion and vehicle or ATM inhibitor (KU55933) treatment (n = 10 in WT vehicle group; n = 10 in WT Ang II + vehicle group; n = 9 in HMGB1-Tg Ang II + vehicle group; n = 7 in WT Ang II + KU55933 group; n = 9 in HMGB1-Tg Ang II + KU55933 group). (D) Analysis of cardiac fibrosis by Masson’s trichrome staining in left ventricular sections in WT and HMGB1-Tg mice subjected to Ang II infusion and vehicle or ATM inhibitor (KU55933) treatment (n = 6 per group). (E) Representative images of CM cross-sectional area by H&E staining (Scale bars = 20 μm) and cardiac fibrosis…