SIRT3 deacetylates and activates OPA1 to regulate mitochondrial dynamics during stress

Abstract

Mitochondrial morphology is regulated by the balance between two counteracting mitochondrial processes of fusion and fission. There is significant evidence suggesting a stringent association between morphology and bioenergetics of mitochondria. Morphological alterations in mitochondria are linked to several pathological disorders, including cardiovascular diseases. The consequences of stress-induced acetylation of mitochondrial proteins on the organelle morphology remain largely unexplored. Here we report that OPA1, a mitochondrial fusion protein, was hyperacetylated in hearts under pathological stress and this posttranslational modification reduced the GTPase activity of the protein. The mitochondrial deacetylase SIRT3 was capable of deacetylating OPA1 and elevating its GTPase activity. Mass spectrometry and mutagenesis analyses indicated that in SIRT3-deficient cells OPA1 was acetylated at lysine 926 and 931 residues. Overexpression of a deacetylation-mimetic version of OPA1 recovered the mitochondrial functions of OPA1-null cells, thus demonstrating the functional significance of K926/931 acetylation in regulating OPA1 activity. Moreover, SIRT3-dependent activation of OPA1 contributed to the preservation of mitochondrial networking and protection of cardiomyocytes from doxorubicin-mediated cell death. In summary, these data indicated that SIRT3 promotes mitochondrial function not only by regulating activity of metabolic enzymes, as previously reported, but also by regulating mitochondrial dynamics by targeting OPA1.

FIG 1

Pathological stress induces OPA1 acetylation, which reduces GTPase activity of the enzyme. Immunoblots (IB) showing hyperacetylation of immunoprecipitated (IP) OPA1 during hypertrophy of the heart, induced by angiotensin II (Ang) infusion for 30 days (A) or by transthoracic aortic constriction (TAC) for 6 months (B), and from hearts of 2-month-old db/db mice (C), compared to respective wild-type (WT) controls. Ac-K, antiacetyllysine antibody; WCL, whole-cell lysates. (D) Reduced GTPase activity of His-OPA1.S1 protein subjected to acetylation (Ac OPA) compared to that of nonacetylated control (CN OPA). GTP hydrolysis (100 μM) was monitored up to 3 h. Values are means ± standard errors of the means (SEM); n = 4; *, P < 0.001. (E) IB showing increased acetylation of OPA1 in SIRT3 knockout (SIRT3KO) hearts compared to WT controls. (F) Bar graph representing GTPase activity for OPA1 immunoprecipitated from immortalized SIRT3-WT and KO MEF lysates. Values are means ± SEM; n = 4; *, P < 0.05. (G) IB showing the amount of OPA1 protein used in the assay illustrated in panel F. IP:IgG is a negative control for IP:OPA1.

FIG 2

SIRT3 binds to and deacetylates OPA1. (A) Autoradiogram (uppermost panel) showing direct binding of His.OPA1 to ³⁵S-labeled SIRT3 but not to SIRT5 in an in vitro binding assay. IBs confirm specificity of indicated proteins. (B) SIRT3 and OPA1 bind to each other when overexpressed in 293T cells. Proteins were immunoprecipitated (IP) using Flag or Myc antibodies and immunoblotted (IB) with indicated antibodies. (C) Corresponding controls for panel B, showing that myc-OPA1 does not cross-react with Flag beads (left panels) or that Flag-SIRT3 does not immunoprecipitate with c-myc (right panels). (D) Endogenous SIRT3 was coprecipitated with OPA1 from WT and not from SIRT3KO hearts. (E) Reduced acetylation of OPA1 immunoprecipitated from HeLa stable cells expressing SIRT3-WT, compared to SIRT3 HY mutant-expressing cells. (F) Reexpression of SIRT3 in SIRT3KO MEFs reduced OPA1 acetylation.

FIG 3

SIRT3 regulates GTPase activity of OPA1 and mitochondrial fusion. (A) Bar diagram showing GTPase activity of acetylated OPA1, compared to SIRT3-mediated deacetylated OPA1, as assessed by an in vitro enzymatic assay. GTP hydrolysis by His-OPA1.S1 protein was compared for indicated pretreatments: nonacetylated (OPA1+PCAF), acetylated (OPA1+PCAF+AcCoA), deacetylated (Ac.OPA1+SIRT3+NAD⁺), and subjected to deacetylation in the absence of NAD (Ac.OPA1+SIRT3 − NAD⁺). Bars represent means ± SEM, n = 4. *, P < 0.001. (B) Immunoblot shows acetylation status of OPA1 and an equal amount of protein used for the activity assay. (C) Reduced mitochondrial fusion in adult SIRT3KO cardiac fibroblasts as measured by diffusion of mtPA-GFP to neighboring mitochondria, compared to WT controls. Total mitochondrial population was visualized by mtDSRed2 expression. Scale bar, 10 μm. (D) Quantitation for mitochondrial networking factor (MNF) from the data acquired in the experiment shown in panel C. Means ± SEM are shown, n = 20 events each. AU, arbitrary units; *, P < 0.001.

FIG 4

SIRT3KO cardiac fibroblasts exhibit loss of mitochondrial membrane potential (ΔΨm). (A) Still images over a time period of 5 min of WT and SIRT3KO cardiac fibroblasts expressing mtGFP (green) and colabeled with TMRM (red, appearing yellow when merged with green). In live videography, lack and delay in the appearance of yellow mitochondria indicate a defect in ΔΨm. (B) Quantitation of depolarized mitochondria (ratio of TMRM-positive to total mitochondria) for the images shown in panel A. The data shown are representative of four separate experiments. Scale bar, 5 μm.

FIG 5

Oxygen consumption rate (OCR) for stable cells expressing OPA1 K mutants in OPA1-null MEFs. (A) Schematic representation of the OPA1 showing locations of acetylated K 926 and K931 residues in the C-terminal GED region. (B) IB showing expression levels of OPA1 mutants in OPA1-null MEFs. (C) Bar diagram depicting basal OCR for immortalized OPA1 WT and OPA1-null MEFs. Values are means ± SEM; n = 3; *, P < 0.001. (D) OPA1-null MEFs were expressed with retroviral vectors (r) synthesizing WT or OPA1 K-to-R mutants. Bar diagram shows basal OCR for different cells. Values are means ± SEM; n = 3; *, P < 0.001. (E and F) Basal OCR of cells expressing K926 and K931 mutated to R or Q. Both K926Q and K931Q (acetylated form) have significantly lower OCR than their respective R mutants. Bars represent means ± SEM; n = 4; *, P < 0.005.

FIG 6

Transmission electron micrographs showing mitochondrial morphology in SIRT3KO hearts. Electron micrographs showing mitochondrial morphology and arrangement in adult wild-type (WT) (A and C) and SIRT3KO hearts (B and D). SIRT3KO micrographs show mitochondria clustered in islands (B). Arrows in SIRT3KO micrographs (D) indicate defective mitochondrial inner membrane fusion. M, mitochondria; S, sarcomeres. Scale bars: 2 μm (A and B); 1 μm (C [left panel] and D [left top panel]); and 200 nm (C [right panel] and D [bottom and right top panels]).

FIG 7

SIRT3 overexpression protects cells from doxorubicin-induced mitochondrial destruction. (A) Representative confocal images of cardiomyocytes showing progressive deterioration of mitochondria when treated with increasing doses of doxorubicin (Dox). Mitochondria are visualized by overexpressing cells with mito-GFP (green, upper panel). SIRT3 overexpression protected the mitochondrial morphology of Dox-treated cardiomyocytes (red, lower panel). Scale bar, 5 μm. Insets display the mitochondrial morphology in an enlarged image in each panel. (B) Unlike stable HeLa cells expressing SIRT3 catalytic mutant (bottom panel) or the vector (top panel), mitochondrial morphology was preserved in SIRT3 WT-expressing cells when treated with Dox (middle panel). Insets show mitochondrial morphology in enlarged images. Scale bar, 1 μm.

FIG 8

SIRT3 expression protects cells from doxorubicin-mediated cell death. (A) Cell death monitored by TUNEL (green) staining for cardiomyocytes overexpressing either adenoviral vector (Ad.Vector) or SIRT3-adenovirus (Ad.SIRT3) and treated with doxorubicin or vehicle for 24 h. Nuclei are marked with DAPI staining (blue). Scale bar, 10 μm. (B) Quantification of TUNEL-positive cells shown in panel A. Values are means ± SEM, n = 4. *, P < 0.001. (C) IB showing acetylated OPA1 immunoprecipitated from neonatal rat cardiomyocytes treated with 300 nM doxorubicin. The right lane shows that overexpressed SIRT3 binds to and deacetylates endogenous OPA1. Veh., vehicle; vect., vector.