Declining NAD(+) induces a pseudohypoxic state disrupting nuclear-mitochondrial communication during aging

Abstract

Ever since eukaryotes subsumed the bacterial ancestor of mitochondria, the nuclear and mitochondrial genomes have had to closely coordinate their activities, as each encode different subunits of the oxidative phosphorylation (OXPHOS) system. Mitochondrial dysfunction is a hallmark of aging, but its causes are debated. We show that, during aging, there is a specific loss of mitochondrial, but not nuclear, encoded OXPHOS subunits. We trace the cause to an alternate PGC-1α/β-independent pathway of nuclear-mitochondrial communication that is induced by a decline in nuclear NAD(+) and the accumulation of HIF-1α under normoxic conditions, with parallels to Warburg reprogramming. Deleting SIRT1 accelerates this process, whereas raising NAD(+) levels in old mice restores mitochondrial function to that of a young mouse in a SIRT1-dependent manner. Thus, a pseudohypoxic state that disrupts PGC-1α/β-independent nuclear-mitochondrial communication contributes to the decline in mitochondrial function with age, a process that is apparently reversible.

Figure 1. Aging and Loss of SIRT1 Leads to a Specific Decline in Mitochondrial-Encoded Genes and Impairment in Mitochondrial Homeostasis in Skeletal Muscle

(A) ATP content of 6-, 22-, and 30-month-old mice (n = 5, *p < 0.05 versus 6-month-old mice). (B) Cytochrome c oxidase (COX) activity (n = 5, *p < 0.05 versus 6-month-old animals). (C and D) Mitochondrial DNA content (C) and DNA integrity (D) (n = 5, *p < 0.05 versus 6-month-old animals). (E) Expression of nuclear- and mitochondrially encoded genes (n = 5, *p < 0.05 versus 6-month-old animals). (F) Immunoblot for COX2 and COX4 in 6-, 22-, and 30-month-old mice. (G) Expression of nuclear- (NDUFS8, NDUFAS, SDHb, SDHd, Uqcrc1, Uqcrc2, COX5b, Cox6a1, ATP5a1, and ATPc1) and mitochondrially encoded genes (ND1, ND2, ND3, ND4, ND4l, ND5, ND6, Cytb, COX1, COX2, COX3, ATP6, and ATP8) in WT and SIRT1 iKO mice (n = 5, *p < 0.05 versus WT). (H and I) (H) Immunoblot for COX2 and COX4 and (I) ATP content in WT and SIRT1 iKO mice (n = 5, *p < 0.05 versus WT). (J) Mitochondrial DNA content of WT and SIRT1 iKO mice (n = 5, *p < 0.05 versus WT). (K) Electron microscopy of gastrocnemius from WT and SIRT1 iKO mice and mitochondrial area (n = 4). (L) Expression of nuclear- and mitochondrially encoded genes in SIRT1 flox/flox Cre-ERT2 primary myoblasts treated with vehicle (0 hr) or tamoxifen (OHT) to induce SIRT1 excision for 6, 12, 24, and 48 hr (n = 4, *p < 0.05 versus vehicle). (M) Mitochondrial mass by NAO fluorescence in SIRT1 flox/flox Cre-ERT2 primary myoblasts treated with vehicle (0 hr) or OHT to induce SIRT1 excision for 6, 12, 24, and 48 hr (n = 4, *p < 0.05 versus vehicle). Nuclear- and mitochondrially encoded genes were ND1, Cytb, COX1, ATP6 and NDUFS8, SDHb, Uqcrc1, COX5b, ATP5a1, respectively. Tissue samples are gastrocnemius unless otherwise stated. Values are expressed as mean ± SEM. See also Figure S1.

Figure 2. Nuclear NAD⁺ Levels Regulate Mitochondrial-Encoded Genes and Mitochondrial Homeostasis through SIRT1, Independently of PGC-1α/β

(A) NAD⁺ levels in gastrocnemius of 6-, 22-, and 30-month-old mice (n = 5, *p < 0.05 versus 6-month-old mice). (B–D) Expression of nuclear- and mitochondrially encoded genes in primary myoblasts transduced with NMNAT1 (B), NMNAT2 (C), NMNAT3 (D), or nontargeting shRNA (n = 4, *p < 0.05 versus shNT). (E and F) Mitochondrial DNA content and (H) ATP content (I) in primary myoblasts transduced with NMNAT1 or nontargeting shRNA (n = 4, *p < 0.05 versus shNT). (G) Expression of mitochondrially encoded genes in tibialis of 10- to 12-month-old mice overexpressing NMNAT1 compared to the contralateral tibialis muscle treated with vehicle (n = 4, *p < 0.05 versus vehicle). (H) Expression of mitochondrially encoded genes in SIRT1 flox/flox Cre-ERT2 primary myoblasts treated with vehicle or OHT to induce SIRT1 excision infected with adenovirus overexpressing NMNAT1 or empty vector (n = 4, *p < 0.05 versus vehicle empty vector). (I and J) Expression of nuclear- and mitochondrially encoded genes in WT and PGC-1α/β knockout myotubes treated with adenovirus overexpressing SIRT1 (I) or NMNAT1 (J) (n = 4, *p < 0.05 versus WT empty; #p < 0.05 versus PGC-1α/β KO empty). Nuclear- and mitochondrially encoded genes were ND1, Cytb, COX1, ATP6 and NDUFS8, SDHb, Uqcrc1, COX5b, ATP5a1, respectively. Tissue samples are gastrocnemius muscle unless otherwise stated. Values are expressed as mean ± SEM. See also Figure S2.

Figure 3. Loss of SIRT1 Induces a Pseudohypoxic State that Disrupts Mitochondrial-Encoded Genes and Mitochondrial Homeostasis

(A and B) HK2, PFKM, PKM, and LDHA mRNA (A) and lactate levels (B) of WT and SIRT1 iKO mice (n = 5, *p < 0.05 versus WT). (C) Immunoblot for HIF-1α and tubulin in WT and SIRT1 iKO mice and in SIRT1 flox/flox Cre-ERT2 primary myoblasts treated with vehicle or OHT to induce SIRT1 excision for 24 hr (SIRT1 iKO). (D) Immunoblot for HIF-1α and tubulin in primary myoblasts transduced with NMNAT1 or nontargeting shRNA. (E) Immunoblot of HIF-1α and tubulin in primary myoblasts treated with pyruvate, lactate, or vehicle for 24 hr. (F) Immunoblot for HIF-1α and tubulin in WT and EglN1 KO mice. (G) Expression of nuclear- and mitochondrially encoded genes of WT and EglN1 KO mice (n = 5, *p < 0.05 versus WT). (H) Mitochondrial DNA content of WT and EglN1 KO mice (n = 5, *p < 0.05 versus WT). (I) Expression of mitochondrially encoded genes in PGC-1α/β KO myotubes treated with adenovirus overexpressing SIRT1 treated with DMSO or the HIF-stabilizing compound DMOG (n = 4, *p < 0.05 versus empty DMSO; #p < 0.05 versus SIRT1 OE DMSO). (J) Immunoblot for HA tag and tubulin in control and C2C12 cells overexpressing either HIF-1α or HIF-2α with the proline residues mutated (HIF-1α DPA and HIF-2α DPA). (K) Expression of nuclear- versus mitochondrially encoded genes in HIF-1α DPA or HIF-2α DPA C2C12 cells (n = 6, *p < 0.05 versus empty vector). (L) Expression of mitochondrially encoded genes in HIF-1α DPA or HIF-2α DPA C2C12 cells with adenovirus overexpressing SIRT1 (n = 4, *p < 0.05 versus empty vector; #p < 0.05 versus SIRT1 OE). (M) Immunoblot for HIF-1α and tubulin in SIRT1 flox/flox Cre-ERT2 primary myoblasts transduced with HIF-1α or nontargeting shRNA and treated with DMOG. (N) Mitochondrial DNA in SIRT1 flox/flox Cre-ERT2 primary myoblasts transduced with HIF-1α or nontargeting shRNA, treated with vehicle or OHT to induce SIRT1 excision (SIRT1 iKO) (n = 4, *p < 0.05 versus shNT vehicle; #p < 0.05 versus shNT SIRT1 iKO). (O) Expression of mitochondrially encoded genes in SIRT1 flox/flox Cre-ERT2 primary myoblasts transduced with HIF-1α or nontargeting shRNA and treated with OHT to induce SIRT1 excision (SIRT1 iKO) (n = 4, *p < 0.05 versus shNT vehicle; #p < 0.05 versus shNT SIRT1 iKO). (P) ATP content in SIRT1 flox/flox Cre-ERT2 primary myoblasts transduced with HIF-1α or nontargeting shRNA and treated with vehicle or OHT to induce SIRT1 excision (n = 5, *p < 0.05 versus shNT vehicle; #p < 0.05 versus shNT SIRT1 iKO). Nuclear- and mitochondrially encoded genes were ND1, Cytb, COX1, ATP6 and NDUFS8, SDHb, Uqcrc1, COX5b, ATP5a1, respectively. Tissue samples are gastrocnemius unless otherwise stated. Values are expressed as mean ± SEM. See also Figure S3.

Figure 4. SIRT1 Regulates HIF-1α Stabilization in Skeletal Muscle through Regulation of VHL Expression

(A and B) Immunoblot for VHL and tubulin in gastrocnemius of WT and SIRT1 iKO mice (A) and WT and SIRT1-Tg mice (B). (C and D) VHL mRNA in WT and SIRT1 iKO mice (C) and WT and SIRT1-Tg mice (D). Values normalized to WT mice (n = 5, *p < 0.05 versus WT). (E) Immunoblot for VHL, HIF-1α, and tubulin in gastrocnemius of 6- and 22-month-old mice. (F) VHL promoter activity in SIRT1 flox/flox Cre-ERT2 primary myoblasts treated with vehicle or OHT for 24 hr to induce SIRT1 excision (SIRT1 iKO). Luciferase values normalized to vehicle cells (n = 5). (G) VHL promoter activity in primary myoblasts with adenovirus expressing SIRT1 or empty vector. Luciferase values normalized to empty vector cells (n = 5). (H) Immunoblot for VHL and tubulin in SIRT1 flox/flox Cre-ERT2 primary myoblasts transduced with VHL or nontargeting shRNA. (I) Representative immunoblot for HIF-1α in SIRT1 flox/flox Cre-ERT2 primary myoblasts transduced with VHL or nontargeting shRNA and treated with OHT for 24 hr (SIRT1 iKO), after which SIRT1 was added back by adenoviral infection. (J) Expression of mitochondrially encoded genes in primary myoblasts transduced with VHL or nontargeting shRNA and treated with adenovirus expressing SIRT1 or empty vector (n = 5, *p < 0.05 versus shNT empty; #p < 0.05 versus shNT SIRT1 OE). Mitochondrially encoded genes were ND1, Cytb, COX1, and ATP6. Values are expressed as mean ± SEM. See also Figure S4.

Figure 5. SIRT1 Regulates Mitochondrial Homeostasis by Modulation of the TFAM Promoter through HIF-1α/c-Myc

(A) TFAM mRNA analyzed by qPCR in gastrocnemius of WT and SIRT1 iKO animals. Values were normalized to WT mice (n = 5, *p < 0.05 versus WT). (B) TFAM promoter activity in SIRT1 flox/flox Cre-ERT2 primary myoblasts treated with vehicle or OHT to induce SIRT1 excision for 24 hr (SIRT1 iKO). Relative luciferase values were normalized to vehicle cells (n = 6, *p < 0.05 versus vehicle). (C) Immunoblot for SIRT1, TFAM, and tubulin in SIRT1 flox/flox Cre-ERT2 primary myoblasts treated with vehicle or OHT to induce SIRT1 excision (SIRT1 iKO) for 24 or 48 hr, after which cells were infected with control or TFAM adenovirus. (D) Expression of nuclear- versus mitochondrially encoded genes in SIRT1 flox/flox Cre-ERT2 primary myoblasts treated with OHT to induce SIRT1 excision (SIRT1 iKO) for 24 or 48 hr, after which cells were infected with TFAM adenovirus. Values were normalized to vehicle cells (n = 4, *p < 0.05 versus vehicle; #p < 0.05 versus SIRT1 iKO 24 hr; &p < 0.05 versus SIRT1 iKO 48 hr). (E) ATP content in SIRT1 flox/flox Cre-ERT2 primary myoblasts treated with vehicle or OHT for 24 or 48 hr as in (D) (n = 4, *p < 0.05 versus vehicle; #p < 0.05 versus SIRT1 iKO 24 hr; &p < 0.05 versus SIRT1 iKO 48 hr). (F) Immunoblot of SIRT1, VHL, HIF-1α, TFAM, and tubulin in SIRT1 flox/flox Cre-ERT2 primary myoblasts treated with vehicle or OHT to induce SIRT1 excision and in cells treated with OHT for 24 hr, after which SIRT1 was added back by adenoviral infection. (G) Interaction of HIF-1α and c-Myc determined by immunoprecipitation of HIF-1α in SIRT1 flox/flox Cre-ERT2 primary myoblasts treated with OHT to excise SIRT1. (H) The c-Myc-binding site on the TFAM promoter. (I) TFAM promoter activity in primary myoblasts transduced with c-Myc or nontargeting shRNA (n = 4, *p < 0.05 versus shNT). (J–L) TFAM promoter full-length activity or with mutation of c-Myc-binding site (Δ c-Myc) in primary myoblasts overexpressing c-Myc (J), PGC-1α (K), SIRT1 (L), or empty vector (n = 4, *p < 0.05 versus empty; #p < 0.05 versus Δ c-Myc empty). (M and N) Chromatin immunoprecipitation (ChIP) (M) and respective quantification by qPCR (N) of c-Myc and HIF-1α to the TFAM promoter in SIRT1 flox/flox Cre-ERT2 primary myoblasts treated with vehicle or OHT to induce SIRT1 excision (n = 3, *p < 0.05 versus vehicle). (O) ChIP of c-Myc to the TFAM promoter in SIRT1 flox/flox Cre-ERT2 primary myoblasts transduced with HIF-1α or nontargeting shRNA treated with vehicle or OHT to induce SIRT1 excision for 24 hr (SIRT1 iKO). Mitochondrially encoded genes were ND1, Cytb, COX1, ATP6. Values are expressed as mean ± SEM. See also Figure S5.

Figure 6. AMPK Activity Regulates Switch between PGC-1α-Dependent and -Independent Mechanisms of Mitochondrial Regulation by SIRT1

(A) Immunoblot for p-AMPK (Thr172) and AMPK in SIRT1 flox/flox Cre-ERT2 primary myoblasts treated with vehicle (0 hr) or OHT to induce SIRT1 excision. (B) Immunoblot for p-ACC (Ser79) and ACC in SIRT1 flox/flox Cre-ERT2 primary myoblasts treated with vehicle or OHT for 48 hr (SIRT1 iKO) and infected with AMPK-DN adenovirus. (C) Expression of nuclear- and mitochondrially encoded genes as in SIRT1 flox/flox Cre-ERT2 primary myoblasts treated with vehicle or OHT (SIRT1 iKO) and infected with empty or AMPK-DN adenovirus for the same period of time (n = 4, *p < 0.05 versus vehicle; #p < 0.05 versus SIRT1 iKO). (D) Immunoblot for p-AMPK (Thr172) and AMPK in SIRT1 flox/flox Cre-ERT2 primary myoblasts treated with vehicle or OHT for 24 or 48 hr, after which cells were infected with control or TFAM adenovirus. (E) Immunoblot for p-AMPK (Thr172) and AMPK in gastrocnemius of WT and SIRT1 iKO mice under fed and fasted conditions. (F) Representative immunoblot for p-AMPK (Thr172) and AMPK in gastrocnemius of 6- and 22-month-old mice. Nuclear- and mitochondrially encoded genes were ND1, Cytb, COX1, ATP6, and NDUFS8, SDHb, Uqcrc1, COX5b, and ATP5a1, respectively. Values are expressed as mean ± SEM.

Figure 7. Increasing NAD⁺ Levels Rescues Age-Related Pseudohypoxia and OXPHOS Dysfunction through a SIRT1-HIF-1α Pathway

(A) Immunoblot for HIF-1α and tubulin of 6- and 22-month-old AL and 22-month-old CR mice. (B) NAD⁺ levels in the same cohorts as in (A) (n = 5, *p < 0.05 versus 6-month-old animals; #p < 0.05 versus 22-month-old AL). (C) Expression of mitochondrially encoded genes of the same cohorts as in (A). Values normalized to 6-month-old mice (n = 5, *p < 0.05 versus 6-month-old mice; #p < 0.05 versus 22-month-old AL). (D) Cytochrome c oxidase (COX) activity (n = 4, *p < 0.05 versus 6-month-old animals; #p < 0.05 versus 22-month-old AL). (E) NAD⁺ levels in 6- and 22-month-old mice treated with vehicle (PBS) or NMN (n = 6, *p < 0.05 versus 6-month-old PBS; #p < 0.05 versus 22-month-old PBS). (F) Immunoblot for VHL, HIF-1α, and tubulin of same cohorts as in (E). (G) Lactate levels of same cohorts as in (E) (n = 6, *p < 0.05 versus 6-month-old PBS; #p < 0.05 versus 22-month-old PBS). (H) Expression of mitochondrially encoded genes of same cohorts as in (E). Values normalized to 6-month-old PBS mice (n = 6, *p < 0.05 versus 6-month-old PBS; #p < 0.05 versus 22-month-old PBS). (I) ATP content of same cohorts as in (E) (n = 6, *p < 0.05 versus 6-month-old PBS; #p < 0.05 versus 22-month-old PBS). (J) Expression of mitochondrially encoded genes in WT and EglN1 KO mice treated with either vehicle (PBS) or NMN (n = 5, *p < 0.05 versus WT PBS). (K) ATP content of same cohorts as in (J) (n = 5, *p < 0.05 versus WT PBS). (L) Expression of mitochondrially encoded genes of WT and SIRT1 iKO mice treated with either vehicle (PBS) or NMN (n = 4, *p < 0.05 versus WT PBS). (M) Expression of mitochondrially encoded genes in primary myoblasts transduced with NMNAT1 or nontargeting shRNA treated with either PBS or NMN (n = 4, *p < 0.05 versus shNT vehicle). (N) Nuclear-mitochondrial communication and its decline during aging. Nuclear NAD⁺ levels regulate mitochondria via a PGC-1α-independent pathway that ensures the correct stoichiometry of OXPHOS subunits, but over time, a chronic pseudohypoxic response is activated, inhibiting OXPHOS. Mitochondrially encoded genes were ND1, Cytb, COX1, and ATP6. Values are expressed as mean ± SEM. See also Figure S6.