SIRT3 deficiency exacerbates ischemia-reperfusion injury: implication for aged hearts

Abstract

Ischemia-reperfusion (IR) injury is significantly worse in aged hearts, but the underlying mechanisms are poorly understood. Age-related damage to mitochondria may be a critical feature, which manifests in an exacerbation of IR injury. Silent information regulator of transcription 3 (SIRT3), the major mitochondrial NAD(+)-dependent lysine deacetylase, regulates a variety of functions, and its inhibition may disrupt mitochondrial function to impact recovery from IR injury. In this study, the role of SIRT3 in mediating the response to cardiac IR injury was examined using an in vitro model of SIRT3 knockdown (SIRT3(kd)) in H9c2 cardiac-derived cells and in Langendorff preparations from adult (7 mo old) wild-type (WT) and SIRT3(+/-) hearts and aged (18 mo old) WT hearts. SIRT3(kd) cells were more vulnerable to simulated IR injury and exhibited a 46% decrease in mitochondrial complex I (Cx I) activity with low O2 consumption rates compared with controls. In the Langendorff model, SIRT3(+/-) adult hearts showed less functional recovery and greater infarct vs. WT, which recapitulates the in vitro results. In WT aged hearts, recovery from IR injury was similar to SIRT3(+/-) adult hearts. Mitochondrial protein acetylation was increased in both SIRT3(+/-) adult and WT aged hearts (relative to WT adult), suggesting similar activities of SIRT3. Also, enzymatic activities of two SIRT3 targets, Cx I and MnSOD, were similarly and significantly inhibited in SIRT3(+/-) adult and WT aged cardiac mitochondria. In conclusion, decreased SIRT3 may increase the susceptibility of cardiac-derived cells and adult hearts to IR injury and may contribute to a greater level of IR injury in the aged heart.

Keywords: SIRT3; acetylation; aging; heart; ischemia; mitochondria; reperfusion.

Fig. 1.

Silent information regulator of transcription 3 knockdown (SIRT3^kd) exacerbates simulated ischemia-reperfusion (IR) injury and changes cellular bioenergetics. SIRT3 protein was knocked down in H9c2 cardiac-derived cells. Seventy-two hours post-transfection, cells were harvested for Western blot analysis and functional assays. A: SIRT3 and β-actin expressions in control (Ctrl) vs. SIRT3^kd cells. A blot image representative of 5 independent experiments is shown. B: lactate dehydrogenase (LDH) release from Ctrl and SIRT3^kd cells during simulated IR injury. LDH release is expressed as a percentage of total LDH. C: NADH dehydrogenase (ubiquinone) 1 α subcomplex subunit 9 (NDUFA9) and β-actin expressions in Ctrl vs. SIRT3^kd cells. D: complex I (Cx I) activity in Ctrl vs. SIRT3^kd cells. E: changes in O₂ consumption rate (OCR) upon injections of FCCP (500 nM) and antimycin A (AA; 5 μM). OCR was measured in Ctrl vs. SIRT3^kd cells in the XF24 Flux Analyzer (Seahorse Bioscience, Billerica, MA). All data are shown as means ± SE, n = 4–5 runs; *P < 0.05 vs. Ctrl using t-test.

Fig. 2.

IR injury in wild-type (WT) adult vs. SIRT3^+/− adult vs. WT aged hearts. Three groups of animals were tested: WT adult (7 mo), SIRT3^+/− adult (7 mo), and WT aged (18 mo). All hearts were subjected to IR, comprising 25 min ischemia and 1 h reperfusion (see methods for details). A: changes in the rate pressure products during IR (presented as percentages of pre-ischemic levels). B: myocardial infarction after IR injury. Top: individual data points for each condition are shown on the left, with average quantified infarct/whole heart ratios on the right. Bottom: typical cross-sections of hearts stained for infarct (white) and viable tissue (red). All data are shown as means ± SE, n = 9 for adult WT, n = 7 for adult SIRT3^+/−, and n = 12 for aged WT; *P < 0.05 vs. adult WT (B); *significance for both SIRT3^+/− adult and WT aged groups vs. WT adults (A).

Fig. 3.

Mitochondrial protein expression and acetylation in WT adult vs. SIRT3^+/− adult vs. WT aged hearts. Mitochondria were isolated and lysed for Western blotting, as detailed in methods. The same blot was probed with antibodies indicated in the pictures. A: mitochondrial SIRT3 and voltage-dependent anion channel (VDAC) expressions before ischemia. SIRT3/VDAC ratio is shown below the blot. B: global lysine acetylation (K-Ac) of mitochondrial samples before ischemia. Right: corresponding densitometry profiles of the acetylation lysine (Ac-Lys) signal intensity in the 20- to 150-kDa range. Bottom: Ac-Lys densitometry in the 37- to 50-kDa range, normalized to protein across the same molecular mass range (delineated with the dotted rectangle) from Ponceau S-stained membranes (see C). C: corresponding Ponceau S-stained membrane (loading control for A and B). D: mitochondrial SIRT3 and VDAC expressions after IR injury. E: global K-Ac of mitochondrial samples after IR injury. F: corresponding Ponceau S-stained membrane (loading control for D and E). Data are shown as means ± SE, n = 3 for pre-ischemic immunoblots, and n = 4 for IR injuries; *P < 0.05 vs. WT adult (ANOVA).

Fig. 4.

Cx I and MnSOD enzymatic activities in mitochondria from WT adult vs. SIRT3^+/− adult vs. WT aged hearts. Mitochondria were isolated from nonischemic heart (Pre-IR) and at the end of the reperfusion protocol (Post-IR). For enzymatic assays, frozen mitochondria were used; data expressed as percent of adult WT. A: Cx I activity Pre-IR. B: MnSOD activity Pr-IR. C: Cx I activity Post-IR. D: MnSOD activity Post-IR. Data are shown as means ± SE, n = 3 for Pre-IR, and n = 4 for Post-IR; *P < 0.05 vs. WT adult (ANOVA).