Abstract
Sirt6, a class III NAD+-dependent deacetylase of the sirtuin family, is a highly specific H3 deacetylase and plays important roles in regulating cellular growth and death. The induction of oxidative stress and death is the critical mechanism involved in cardiomyocyte injury and cardiac dysfunction in doxorubicin-induced cardiotoxicity, but the regulatory role of Sirt6 in the fate of DOX-impaired cardiomyocytes is poorly understood. In the present study, we exposed Sirt6 heterozygous (Sirt6+/−) mice and their littermates as well as cultured neonatal rat cardiomyocytes to DOX, then investigated the role of Sirt6 in mitigating oxidative stress and cardiac injury in the DOX-treated myocardium. Sirt6 partial knockout or silencing worsened cardiac damage, remodeling, and oxidative stress injury in mice or cultured cardiomyocytes with DOX challenge. Cardiomyocytes infected with adenoviral constructs encoding Sirt6 showed reversal of this DOX-induced damage. Intriguingly, Sirt6 reduced oxidative stress injury by upregulating endogenous antioxidant levels, interacted with oxidative stress-stirred p53, and acted as a co-repressor of p53 in nuclei. Sirt6 was recruited by p53 to the promoter regions of the target genes Fas and FasL and further suppressed p53 transcription activity by reducing histone acetylation. Sirt6 inhibited Fas/FasL signaling and attenuated both Fas-FADD-caspase-8 apoptotic and Fas-RIP3 necrotic pathways. These results indicate that Sirt6 protects the heart against DOX-induced cardiotoxicity by upregulating endogenous antioxidants, as well as suppressing oxidative stress and cell death signaling pathways dependent on ROS-stirred p53 transcriptional activation, thus reducing Fas–FasL-mediated apoptosis and necrosis.
Graphical abstract
•Sirt6 is significantly decreased in DOX-insulted mouse hearts and cardiomyocytes.
•Sirt6 attenuates DOX-induced cardiac atrophy, dysfunction and oxidative stress.
• Sirt6 reduces oxidative stress injury by upregulating endogenous antioxidants.
• Sirt6 interacts with p53 as a co-repressor to suppress p53 transcriptional regulation and inhibits Fas-FasL–mediated apoptosis and necrosis downstream of p53.
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The authors declare that all materials and data generated or analyzed in this study are available within this article and the supplementary materials.
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Abbreviations
- CK:
-
Creatine kinase
- CK-MB:
-
Creatine kinase-MB
- CO:
-
Cardiac output
- DMSO:
-
Dimethyl sulfoxide
- DOX:
-
Doxorubicin
- dPmax :
-
Maximal value of the first derivative of LV pressure
- dPmin :
-
Minimal value of the first derivative of LV pressure
- EDV:
-
End-diastolic volume
- EF:
-
Ejection fraction
- ESV:
-
End-systolic volume
- FADD:
-
Fas associated via death domain
- Fas:
-
Tumor necrosis factor receptor superfamily member 6
- FasL:
-
Fas receptor-Fas ligand
- FS:
-
Fractional shortening
- GFP:
-
Green fluorescent protein
- Gpx1:
-
Glutathione peroxidase 1
- H3K9Ac:
-
Acetylation on histone H3 lysine 9
- HMGB1:
-
High mobility group box 1
- KO:
-
Knockout
- LDH:
-
Lactate dehydrogenase
- LV:
-
Left ventricular
- LVEDD:
-
LV end diastolic diameter
- LVEDV:
-
LV end-diastolic volume
- LVEDP:
-
LV end-diastolic pressure
- LVESP:
-
LV end-systolic pressure
- LVIDs:
-
LV internal dimension in systole
- LVIDd:
-
LV internal dimension in diastole
- LVP:
-
LV pressure
- LVPWd:
-
LV posterior wall thickness at end diastole
- LVSd:
-
Left ventricle systolic diameter
- LVW/TL:
-
Left heart ventricle weight/tibia length
- NRCMs:
-
Neonatal rat cardiomyocytes
- Prx5:
-
Peroxiredoxin 5
- ROS:
-
Reactive oxygen species
- SOD2:
-
Mn-dependent superoxide dismutase 2
- SV:
-
Stroke volume
- WT:
-
Wild type
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Acknowledgements
Special thanks to Laura Smales for the help in editing and revising the manuscript.
Funding
This work was supported by the National Natural Science Foundation of China (grant nos. 81870221, 81670249, 82070299, 31271226, and 31071001 to Dr. Wei Jiang and 81900276 to Dr. Ruli Li) and Post-Doctor Research Project, West China Hospital, Sichuan University (2019HXBH102 to Dr. Xin Yan and 2020HXBH073 to Dr. Ruli Li).
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SSW, JL, and LYL: investigation, experiments, data collection, and writing original draft. XXW and MMT: idea and formal analysis. LF, YJZ, JYX, XMC, and HYC: validation and formal analysis. XY, RLL, YW, JJX, HL, and XL: animal experiment. KYX and CLZ: provision of materials and instrumentation. WJ: funding acquisition, writing, reviewing, and editing.
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Supplementary information
Fig. S1
Sirt6 was overexpressed in NRCMS via adenovirus infection. NRCMs were infected with control green fluorescent protein adenovirus (Ad-EGFP) or Sirt6 protein adenovirus (Ad-Sirt6) for 48 h; the efficiency of overexpression was assessed by western blot analysis. Data were analyzed by one-way ANOVA. Values represent the mean ± SEM. **P < 0.01 vs Ad-EGFP-treated cells; #P < 0.05 vs Ad-Sirt6-treated cells. (PNG 879 kb)
Fig. S2
Effect of Sirt6 deficiency/overexpression on Bcl2, Bcl-xL, Bax, Bak and phosphorylation of p53 in DOX-treated cardiac tissues and cultured NRCMs. aWild-type or Sirt6+/- mice were treated without or with DOX (4 mg/kg weekly) injection for 4 weeks and maintenance for another one week. Immunoblotting of Bcl-xL, Bcl2, Bax, Bak, p53(phosphor S392), p53 and β-actin with specific antibodies (n = 5/group). b, c NRCMs were transfected with Sirt6 siRNA or NTC or infected with Ad-Sirt6 or Ad-EGFP without or with DOX (2 μM) treatment. Immunoblotting of cell lysates for Bcl-xL, Bcl2, Bax, Bak, p53(phosphor S392), p53 and β-actin with specific antibodies (n = 5/group). All data were analyzed by one-way ANOVA. Values represent the mean ± SEM. In Fig. a: *P < 0.05, **P < 0.01 vs WT control. In Fig. b and c: *P < 0.05, **P < 0.01 vs NTC or Ad-EGFP. (PNG 105 kb)
Fig. S3
Effect of Sirt6 deficiency/overexpression on DOX-induced lactate dehydrogenase (LDH), creatine kinase (CK) and MB isoenzyme of CK (CK-MB) in myocardial tissues or NRCMs. a LDH, CK and CK-MB levels were measured in serum of wild-type(WT) or Sirt6+/- mice without or with DOX (4 mg/kg weekly) for 4 weeks and maintenance for another one week (n= 5/group). *P < 0.05, **P < 0.01 vs WT Control, #P < 0.05 vs DOX-treated WT. b, c LDH, CK and CK-MB levels in cultural supernatants of NTC and Sirt6 siRNA NRCMs or Ad-EGFP and Ad-Sirt6-infected NRCMs without or with 2 μM DOX for 18 h (n = 5/group). All data were analyzed by one-way ANOVA. Values represent the mean ± SEM. In Fig. S2b*P < 0.05, **P < 0.01 vs NTC, #P < 0.05 vs DOX-treated NTC. In Fig. S2c, *P < 0.05, **P < 0.01 vs Ad-EGFP, #P < 0.05 vs DOX-treated Ad-EGFP. (PNG 357 kb)
Fig. S4
Effect of Sirt6 deficiency/overexpression on DOX-induced mRNA expression of peroxiredoxin 5 (Prx5), glutathione peroxidase 1 (Gpx1), catalase (CAT) and Mn-dependent superoxide dismutase 2 (SOD2) in myocardial tissues or NRCMs. a WT or Sirt6+/- mice were treated without or with DOX (4 mg/kg weekly) for 4 weeks and maintenance for another one week. RT-qPCR analysis of relative mRNA levels of the indicated genes, all normalized to β-actin level (n = 5/group). *P < 0.05, **P < 0.01 vs the WT Control, #P < 0.05 vs DOX-treated WT. b, c NTC and Sirt6 siRNA-transfected NRCMs or Ad-EGFP and Ad-Sirt6-infected NRCMs with 2 μM DOX for 18 h or not. RT-qPCR of relative mRNA levels, all normalized to β-actin level (n = 5/group). All data were analyzed by one-way ANOVA. Values represent the mean ± SEM. In Fig. S4b, *P < 0.05, **P < 0.01 vs NTC, #P < 0.05 vs DOX-treated NTC. In Fig. S4c, *P < 0.05, **P < 0.01 vs Ad-EGFP, #P < 0.05 vs DOX-treated Ad-EGFP. (PNG 573 kb)
Fig. S5
Effect of Sirt6 deficiency/overexpression on DOX-induced mRNA expression of p53 transcrption-regulated genes in myocardial tissues or NRCMs. aWild-type or Sirt6+/- mice were treated without or with DOX (4 mg/kg weekly) injection for 4 weeks and maintenance for another one week. RT-qPCR analysis of relative mRNA levels of mouse double minute 2 homolog (Mdm2), cyclin-dependent kinase inhibitor 2A (Cdkn2a), Bcl-2-associated protein X (Bax), Bcl-2antagonist/killer (Bak), TNF receptor superfamily member 6 (Fas), Fas ligand (FasL), phorbol-12-myristate-13-acetate-induced protein 1 (Noxa) and p53 upregulated modulator of apoptosis (Puma), all normalized to β-actin level (n = 5/group). *P < 0.05, **P < 0.01 versus the WT Control group mice, #P < 0.05 versus DOX-treated WT mice. b, c NTC and Sirt6 siRNA-transfected NRCMs, or Ad-EGFP and Ad-Sirt6-infected NRCMs, treated with 2 μM DOX for 18 h or not (n = 5/group). RT-qPCR analysis of relative mRNA levels of Mdm2 and Cdkn2a, Bax, Bak, Fas, FasL, Noxa and Puma, all normalized to β-actin level (n = 5/group). All data were analyzed by one-way ANOVA. Values represent the mean ± SEM. In Fig. S6b *P < 0.05, **P < 0.01 vs NTC, #P < 0.05 vs DOX-treated NTC. In Fig. S6c *P < 0.05, **P < 0.01 vs Ad-EGFP, #P < 0.05 vs DOX-treated Ad-EGFP. (PNG 888 kb)
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Wu, S., Lan, J., Li, L. et al. Sirt6 protects cardiomyocytes against doxorubicin-induced cardiotoxicity by inhibiting P53/Fas-dependent cell death and augmenting endogenous antioxidant defense mechanisms. Cell Biol Toxicol 39, 237–258 (2023). https://doi.org/10.1007/s10565-021-09649-2
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DOI: https://doi.org/10.1007/s10565-021-09649-2