Silent information regulator 1 protects the heart from ischemia/reperfusion

Abstract

Background: Silent information regulator 1 (Sirt1), a class III histone deacetylase, retards aging and protects the heart from oxidative stress. We here examined whether Sirt1 is protective against myocardial ischemia/reperfusion (I/R).

Methods and results: Protein and mRNA expression of Sirt1 is significantly reduced by I/R. Cardiac-specific Sirt1(-/-) mice exhibited a significant increase (44±5% versus 15±5%; P=0.01) in the size of myocardial infarction/area at risk. In transgenic mice with cardiac-specific overexpression of Sirt1, both myocardial infarction/area at risk (15±4% versus 36±8%; P=0.004) and terminal deoxynucleotidyl transferase dUTP nick end labeling-positive nuclei (4±3% versus 10±1%; P<0.003) were significantly reduced compared with nontransgenic mice. In Langendorff-perfused hearts, the functional recovery during reperfusion was significantly greater in transgenic mice with cardiac-specific overexpression of Sirt1 than in nontransgenic mice. Sirt1 positively regulates expression of prosurvival molecules, including manganese superoxide dismutase, thioredoxin-1, and Bcl-xL, whereas it negatively regulates the proapoptotic molecules Bax and cleaved caspase-3. The level of oxidative stress after I/R, as evaluated by anti-8-hydroxydeoxyguanosine staining, was negatively regulated by Sirt1. Sirt1 stimulates the transcriptional activity of FoxO1, which in turn plays an essential role in mediating Sirt1-induced upregulation of manganese superoxide dismutase and suppression of oxidative stress in cardiac myocytes. Sirt1 plays an important role in mediating I/R-induced increases in the nuclear localization of FoxO1 in vivo.

Conclusions: These results suggest that Sirt1 protects the heart from I/R injury through upregulation of antioxidants and downregulation of proapoptotic molecules through activation of FoxO and decreases in oxidative stress.

Figure 1. Sirt1 is downregulated by I/R in vivo

(A) The protocol used for ischemia/reperfusion (I/R) and ischemia preconditioning (IPC) for C57BL/6J mice. (B) Heart homogenates were prepared from mice subjected to sham operation, I/R and IPC. Expression of Sirt1 and tubulin was evaluated by immunoblots. mRNA expression of Sirt1 was evaluated with RT-PCR. The expression level of sham operated mice is expressed as 1.

Figure 2. Myocardial injury caused by I/R is enhanced in cardiac specific Sirt1 -/- mice

Cardiac specific Sirt1 -/- mice and wild type (+/+) mice generated on a C57BL/6J background were subjected to 30 min of ischemia and 24 hours of reperfusion. (A) Gross appearance of LV myocardial sections after Alcian blue and triphenyltetrazolium chloride (TTC) staining. (B) (left) The area at risk (AAR) (% of LV) was comparable between wild type and cardiac specific Sirt1 -/- mice. (right) The infarction area/AAR was significantly greater in cardiac specific Sirt1 -/- mice than in wild type mice.

Figure 3. I/R injury is attenuated in Tg-Sirt1 mice

(A) The protocol used for I/R for Tg-Sirt1 and NTg. Tg-Sirt1 or NTg mice generated on an FVB background were subjected to 45 minutes of ischemia and 24 hours of reperfusion. (B) (upper) Gross appearance of LV myocardial sections after Alcian blue and triphenyltetrazolium chloride (TTC) staining. (lower left) The area at risk (AAR) (% of LV) was comparable between NTg and Tg-Sirt1. (lower right) The infarction area/AAR was significantly smaller in Tg-Sirt1 than in NTg. (C) (left) LV myocardial sections were subjected to TUNEL and DAPI staining. Representative images of the staining in the border zone. (right) The number of TUNEL-positive myocytes was expressed as a percentage of total nuclei detected by DAPI staining.

Figure 4. Myocardial I/R injury is attenuated in Tg-Sirt1 mouse hearts ex vivo

(A) The protocol used for I/R for Tg-Sirt1 and NTg control mice in the Langendorff model. The hearts of Tg-Sirt1 or NTg control mice were subjected to 30 minutes of global ischemia and 60 minutes of reperfusion. (B-F) Hemodynamics of the Langendorff-perfused isolated mouse hearts of Tg-Sirt1 and NTg control mice. (B) LVSP, left ventricular systolic pressure; (C) LVDP, left ventricular developed pressure (systolic pressure – diastolic pressure); (D) LVEDP, left ventricular end-diastolic pressure; (E,F) Systolic and diastolic dP/dt. In B-F, the level at baseline is expressed as 100%. (G) (upper) Gross appearance of LV myocardial sections after triphenyltetrazolium chloride (TTC) staining. (lower) The infarction area/AAR (where AAR= total heart) was significantly smaller in Tg-Sirt1 than in NTg.

Figure 4. Myocardial I/R injury is attenuated in Tg-Sirt1 mouse hearts ex vivo

(A) The protocol used for I/R for Tg-Sirt1 and NTg control mice in the Langendorff model. The hearts of Tg-Sirt1 or NTg control mice were subjected to 30 minutes of global ischemia and 60 minutes of reperfusion. (B-F) Hemodynamics of the Langendorff-perfused isolated mouse hearts of Tg-Sirt1 and NTg control mice. (B) LVSP, left ventricular systolic pressure; (C) LVDP, left ventricular developed pressure (systolic pressure – diastolic pressure); (D) LVEDP, left ventricular end-diastolic pressure; (E,F) Systolic and diastolic dP/dt. In B-F, the level at baseline is expressed as 100%. (G) (upper) Gross appearance of LV myocardial sections after triphenyltetrazolium chloride (TTC) staining. (lower) The infarction area/AAR (where AAR= total heart) was significantly smaller in Tg-Sirt1 than in NTg.

Figure 5. Sirt1 upregulates cardioprotective molecules in the heart

Heart homogenates were prepared from the ischemic area in Tg-Sirt1 and NTg mice subjected to I/R. Expression of Bax, MnSOD, Trx1, Bcl-xL, cleaved caspase-3 and tubulin was evaluated by immunoblots. (A) Representative immunoblots are shown. (B-F) The quantitative and statistical analyses of the immunoblots are shown. The relative expression of MnSOD (B), Trx1 (C), Bcl-xL (D), Bax (E) and cleaved caspase-3 (F) in Tg-Sirt1 versus NTg is shown. The expression level of NTg mice is expressed as 1. (G-I) Representative immunostaining of Bcl-xL (G), cleaved caspase-3 (H), and 8-OHdG (I) in the ischemic area of left ventricular myocardial sections in Tg-Sirt1 and NTg mice subjected to I/R.

Figure 5. Sirt1 upregulates cardioprotective molecules in the heart

Heart homogenates were prepared from the ischemic area in Tg-Sirt1 and NTg mice subjected to I/R. Expression of Bax, MnSOD, Trx1, Bcl-xL, cleaved caspase-3 and tubulin was evaluated by immunoblots. (A) Representative immunoblots are shown. (B-F) The quantitative and statistical analyses of the immunoblots are shown. The relative expression of MnSOD (B), Trx1 (C), Bcl-xL (D), Bax (E) and cleaved caspase-3 (F) in Tg-Sirt1 versus NTg is shown. The expression level of NTg mice is expressed as 1. (G-I) Representative immunostaining of Bcl-xL (G), cleaved caspase-3 (H), and 8-OHdG (I) in the ischemic area of left ventricular myocardial sections in Tg-Sirt1 and NTg mice subjected to I/R.

Figure 6. Sirt1-induced upregulation of MnSOD is mediated by FoxO1

Cultured myocytes were transduced with adenovirus harboring LacZ (control, Ad-LacZ), wild type Sirt1 (Ad-Sirt1-WT), shRNA-Sirt1 (Ad-sh-Sirt1) or tet-inducible p300. Expression of p300 was induced by co-transduction with adenovirus harboring tTA without doxycycline. Forty-eight hours after transduction, cells were harvested. (A) Expression of FoxO1, acetylated FoxO1 and tubulin (internal control) was evaluated with immunoblot analyses. (B,C) The relative expression of FoxO1 and acetylated FoxO1 was evaluated by densitometric analyses. The expression level of FoxO1 and acetylated FoxO1 in Ad-LacZ transduced myocytes is expressed as 1. (D,E) Myocytes were transduced with adenovirus harboring LacZ, wild type FoxO1 (Ad-FoxO1-WT) and shRNA-FoxO1 (Ad-sh-FoxO1). (D) Expression of MnSOD and tubulin (internal control) was evaluated with immunoblot analyses. (E) The relative expression of MnSOD was evaluated by densitometric analyses. The expression level of MnSOD in Ad-LacZ transduced myocytes is expressed as 1. (F) Myocytes were transfected with 3xIRS-Luc and then transduced with Ad-LacZ or Ad-Sirt1 (5 MOI). Ad-Sirt1 transfected myocytes were treated with or without sirtinol (10 μM). Experiments were conducted three times.

Figure 6. Sirt1-induced upregulation of MnSOD is mediated by FoxO1

Cultured myocytes were transduced with adenovirus harboring LacZ (control, Ad-LacZ), wild type Sirt1 (Ad-Sirt1-WT), shRNA-Sirt1 (Ad-sh-Sirt1) or tet-inducible p300. Expression of p300 was induced by co-transduction with adenovirus harboring tTA without doxycycline. Forty-eight hours after transduction, cells were harvested. (A) Expression of FoxO1, acetylated FoxO1 and tubulin (internal control) was evaluated with immunoblot analyses. (B,C) The relative expression of FoxO1 and acetylated FoxO1 was evaluated by densitometric analyses. The expression level of FoxO1 and acetylated FoxO1 in Ad-LacZ transduced myocytes is expressed as 1. (D,E) Myocytes were transduced with adenovirus harboring LacZ, wild type FoxO1 (Ad-FoxO1-WT) and shRNA-FoxO1 (Ad-sh-FoxO1). (D) Expression of MnSOD and tubulin (internal control) was evaluated with immunoblot analyses. (E) The relative expression of MnSOD was evaluated by densitometric analyses. The expression level of MnSOD in Ad-LacZ transduced myocytes is expressed as 1. (F) Myocytes were transfected with 3xIRS-Luc and then transduced with Ad-LacZ or Ad-Sirt1 (5 MOI). Ad-Sirt1 transfected myocytes were treated with or without sirtinol (10 μM). Experiments were conducted three times.

Figure 7. Sirt1 enhances nuclear localization of FoxO1 in response to I/R

NTg, Tg-Sirt1 and cardiac specific Sirt1 -/- mice were subjected to I/R or sham operation. Twenty-four hours after reperfusion, the heart was subjected to immunostaining with anti-troponin T antibody (red), anti-FoxO1 antibody (green) and DAPI (blue). FoxO1 positive nuclei/total nuclei was evaluated and expressed as %.