Regulation of the mPTP by SIRT3-mediated deacetylation of CypD at lysine 166 suppresses age-related cardiac hypertrophy - PubMed (original) (raw)

Angela V Hafner et al. Aging (Albany NY). 2010 Dec.

Abstract

Cardiac failure is a leading cause of age-related death, though its root cause remains unknown. Mounting evidence implicates a decline in mitochondrial function due to increased opening of the mitochondrial permeability transition pore (mPTP). Here we report that the NAD+-dependent deacetylase SIRT3 deacetylates the regulatory component of the mPTP, cyclophilin D (CypD) on lysine 166, adjacent to the binding site of cyclosporine A, a CypD inhibitor. Cardiac myocytes from mice lacking SIRT3 exhibit an age-dependent increase in mitochondrial swelling due to increased mPTP opening, a phenotype that is rescued by cyclosporine A. SIRT3 knockout mice show accelerated signs of aging in the heart including cardiac hypertrophy and fibrosis at 13 months of age. SIRT3 knockout mice are also hypersensitive to heart stress induced by transverse aortic constriction (TAC), as evidenced by cardiac hypertrophy, fibrosis, and increased mortality. Together, these data show for the first time that SIRT3 activity is necessary to prevent mitochondrial dysfunction and cardiac hypertrophy during aging and shed light on new pharmacological approaches to delaying aging and treating diseases in cardiac muscle and possibly other post-mitotic tissues.

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Conflict of interest statement

D. Sinclair is a consultant to and inventor on patents licensed to Sirtris, a GlaxoSmithKline company aiming to develop drugs based on sirtuin modulation.

Figures

Figure 1.

Figure 1.. SIRT3 prevents mitochondrial permeability transition in cardiac tissue during aging.

Mitochondrial swelling induced by Ca2+. Mitochondria from 3 (A), 6 (B) and 16 months old (C) wt and SIRT3−/− mouse hearts subjected to Ca2+-induced mitochondrial swelling, measured as % decrease in the initial optical density (OD540) in the presence or absence of 1 μM CsA (n=4 per group and age). All error bars show s.e.m.

Figure 2.

Figure 2.. Cyclophilin D is acetylated at K166

(A) Representative m/z spectrum of peptide TDWLDG-AcK-HVVFGHVK obtained from mass spectrometry analyses of Flag-purified mouse Cyclophilin D. (B) Sequence of Cyclophilin D (Mus musculus). Location of the identified acetylation site. (Peptide: blue; acetylated Lysine: red). (C) Protein sequence alignment of CypD from Homo sapiens, Mus musculus, Danio rerio, Saccharomyces cerevisiae. Lysine 166 is marked in red.

Figure 3.

Figure 3.. SIRT3 binds to and deacetylates CypD at K166

(A) CypD-K166 lies adjacent to the CsA binding pocket (Protein Data Bank, 2Z6W). (**B)**Interaction studies using HA-tagged SIRT3, SIRT4 and SIRT5 and FLAG-tagged CypD assessed by coimmunoprecipitation. (C) Specificity of a polyclonal antibody raised against acetylated CypD-K166 confirmed by a lack of Western blot signal for mutant CypD-K166R. SIRT3 and SIRT3-H248Y were co-transfected with FLAG-tagged CypD and the level of acetylation at CypD-K166 was assessed. (D) Vectors for FLAG-tagged SIRT3 and SIRT3-H248Y transfected in HEK 293T cells were immunoprecipitated, then incubated with purified CypD in the presence of the SIRT3 co-substrate NAD+.

Figure 4.

Figure 4.. Age- and stress-dependent development of cardiac hypertrophy in SIRT3 −/− mice.

(A) Kaplan-Meier survival plot of 3 months old wt and SIRT3−/− mice after the Transverse Aortic Constriction (TAC) surgery. wt (n=8), SIRT3−/− (n=11). p < 0.05. (B) Representative image and Masson's trichrome staining of hearts from wt and SIRT3−/− mice 4 weeks after the TAC surgery. Heart weight tibia length ratio of wt and SIRT3−/− mice 30 days after the TAC surgery wt (n=8), SIRT3−/− (n=4) mice. (C) Interventricular septal thickness at diastole (Ivs;d) of 2 and 13 month old wt and SIRT3−/− mice after transverse aortic constriction (TAC) for 4 weeks. (D) Representative images of Masson's trichrome staining of transverse sections of heart from 13 month old wt and SIRT3−/− mice. Collagen deposits (fibrosis) stain blue. n=4 mice per group and age (magnification, 20x). **p<0.01. *** p < 0.001. All error bars represent s.e.m.

Figure 5.

Figure 5.. Regulation of the mPTP by SIRT3

In normal cardiac tissue, SIRT3 targets CypD, maintaining it in a deacetylated state, thus preventing opening of the mPTP during aging and induced cardiac stress. In SIRT3−/− mice, however, CypD is hyperacetylated, resulting in increased induction of the mPTP. A decline in SIRT3 activity over time explains the increase in mitochondrial permeability transition and the decline in cardiac function with age. IMS = inner membrane space.

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