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. 2014 Jul 17;10(7):e1004490.
doi: 10.1371/journal.pgen.1004490. eCollection 2014 Jul.

Muscle-specific SIRT1 gain-of-function increases slow-twitch fibers and ameliorates pathophysiology in a mouse model of duchenne muscular dystrophy

Angeliki Chalkiadaki  1 , Masaki Igarashi  1 , Armiyaw Sebastian Nasamu  1 , Jovana Knezevic  1 , Leonard Guarente  1
Affiliations

Muscle-specific SIRT1 gain-of-function increases slow-twitch fibers and ameliorates pathophysiology in a mouse model of duchenne muscular dystrophy

Angeliki Chalkiadaki et al. PLoS Genet. .

Abstract

SIRT1 is a metabolic sensor and regulator in various mammalian tissues and functions to counteract metabolic and age-related diseases. Here we generated and analyzed mice that express SIRT1 at high levels specifically in skeletal muscle. We show that SIRT1 transgenic muscle exhibits a fiber shift from fast-to-slow twitch, increased levels of PGC-1α, markers of oxidative metabolism and mitochondrial biogenesis, and decreased expression of the atrophy gene program. To examine whether increased activity of SIRT1 protects from muscular dystrophy, a muscle degenerative disease, we crossed SIRT1 muscle transgenic mice to mdx mice, a genetic model of Duchenne muscular dystrophy. SIRT1 overexpression in muscle reverses the phenotype of mdx mice, as determined by histology, creatine kinase release into the blood, and endurance in treadmill exercise. In addition, SIRT1 overexpression also results in increased levels of utrophin, a functional analogue of dystrophin, as well as increased expression of PGC-1α targets and neuromuscular junction genes. Based on these findings, we suggest that pharmacological interventions that activate SIRT1 in skeletal muscle might offer a new approach for treating muscle diseases.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. SIRT1 overexpression in skeletal muscle.
(A) Western blot in tissue protein homogenates prepared from WT and transgenic gastrocnemius muscle (line 4140). All following experiments were performed using line Tg-4140, unless otherwise indicated. (B) Gastrocnemius muscle weight (of both hindlimbs) and muscle/body weight ratio of WT and Tg-4140 mice at 10 weeks of age (n = 7–10). (C) Representative H&E staining of gastrocnemius muscle from WT and transgenic mice and quantification of fiber size (arbitrary units) (700–1000 fibers/genotype, n = 3). Data are expressed as mean +/− s.e.m. ***p<0.001 by two-tailed unpaired Student's t test.
Figure 2
Figure 2. SIRT1 overexpression inhibits the expression of muscle atrophy genes.
(A) Relative mRNA levels of MAFBx and MuRF-1 atrophy genes in gastrocnemius muscle of WT and Tg-4140 mice fed or fasted for 24 hrs (n = 3–5). (B) Relative mRNA levels of MAFBx and MuRF-1 atrophy genes in gastrocnemius muscle of WT and Tg-4140 mice, which underwent mock surgery (control) or were denervated for 3 days (n = 3–5). (C) Relative mRNA levels of FOXO1 and FOXO3 transcription factors in gastrocnemius muscle of WT and Tg-4140 mice fed or fasted for 24 hrs (n = 3–5). Data are expressed as mean +/− s.e.m. *p<0.05, **p<0.01, ***p<0.001 by two-tailed unpaired Student's t test.
Figure 3
Figure 3. SIRT1 overexpression drives fast-to-slow fiber type switch.
(A) Relative mRNA levels of troponin slow and troponin fast genes in gastrocnemius muscle of WT and Tg-4140 mice (8–10 weeks old, n = 3–5). (B) Relative mRNA levels of myosin heavy chain 2B, 2x, 2A, and I in gastrocnemius muscle of WT and Tg-4140 mice (n = 3–5). (C) Representative SDH activity staining of cross-sections of gastrocnemius/soleus (upper panels) and gastrocnemius (lower panel) muscle of WT and Tg-4140 mice. Quantitation is shown in Figure S2A. (D) Representative COX activity staining of cross-sections of gastrocnemius muscle of WT and Tg-4140 mice. Quantitation is shown in Figure S2B. (E) Relative mitochondrial DNA of indicated genes normalized to actin (10–12 weeks old, n = 4). (F) Relative mRNA levels of TFAM, PPARα, PPARδ, and PGC-1α in gastrocnemius muscle of WT and Tg-4140 mice (n = 3–5). (G) Relative mRNA levels of mitochondrial-expressed electron transport chain (ETC) genes in gastrocnemius muscle of WT and Tg-4140 mice (10–12 weeks old, n = 3–5). (H) Relative mRNA levels of nuclear-expressed electron transport chain (ETC) genes in gastrocnemius muscle of WT and Tg-4140 mice (10–12 weeks old, n = 3–5). (I) Lysine acetylation levels of PGC-1α (IB: ac-Lys) in protein extracts prepared from gastrocnemius muscle of WT and Tg-4140 and immunoprecipitated by PGC-1α specific antibodies (IP: PGC-1α). Data are expressed as mean +/− s.e.m. *p<0.05, **p<0.01, ***p<0.001 by two-tailed unpaired Student's t test.
Figure 4
Figure 4. SIRT1 overexpression in skeletal muscle does not affect the fasting response.
(A) Blood glucose and plasma insulin levels in fed or after overnight fasting of WT and Tg-4140 mice (8–10 weeks old, n = 6–10). (B–G) Relative mRNA levels of GLUT4, PDK4, mitofusin, mCPT1, ERRα, and MCAD genes in gastrocnemius muscle of fed and fasted WT and Tg-4140 (10–12 weeks old, n = 4). (H) Western blot analyses in gastrocnemius muscle protein extracts prepared from gastrocnemius muscle of WT and Tg-4140 (10–12 weeks old). Data are expressed as mean +/− s.e.m. *p<0.05, **p<0.01, ***p<0.001 by two-tailed unpaired Student's t test.
Figure 5
Figure 5. Loss of SIRT1 from skeletal muscle does not affect fiber type composition but reduces the capacity for endurance exercise.
(A) Western blot in tissue protein homogenates prepared from WT and muscle-specific SIRT1 knockout (MckKO) gastrocnemius muscle. The arrow shows the faster migrating SIRT1 protein lacking the catalytic domain (ΔExon4). Relative RNA levels of SIRT1 transcript using SIRT1 exon 4 specific primers. (B) Gastrocnemius muscle weight (of one hindlimb) of WT and MckKO mice at 10 weeks of age (n = 7–10). (C) Blood glucose and plasma insulin levels in WT and MckKO mice after overnight fasting (8–10 weeks old, n = 6–10). (D) Relative mRNA levels of troponin slow and troponin fast genes in gastrocnemius muscle of WT and MckKO mice (8–10 weeks old, n = 3–5). (E) Relative mRNA levels of myosin heavy chain 2B, 2x, 2A, and I in gastrocnemius muscle of WT and MckKO mice (n = 3–5). (F) Relative mRNA levels of TFAM, PPARα, and PGC-1α in gastrocnemius muscle of WT and MckKO mice (n = 3–5). (G) Distance run in treadmill exercise by WT and MckKO mice (12–14 weeks old, n = 10–12). (H) Relative expression levels of mitochondrial-expressed electron transport chain (ETC) genes (10–12 weeks old, n = 4). (I) Relative expression levels of nuclear-expressed ETC genes (10–12 weeks old, n = 4). Data are expressed as mean +/− s.e.m. *p<0.05, **p<0.01, ***p<0.001 by two-tailed unpaired Student's t test.
Figure 6
Figure 6. SIRT1 overexpression in skeletal muscle of WT and mdx mice induces the expression of neuromuscular junction genes and utrophin.
(A) Western blot in tissue protein homogenates prepared from gastrocnemius muscles of mdx and mdx mice crossed to Tg-4140 (mdx;TG) (B) Body weight, gastrocnemius muscle weight (of both hindlimbs) and muscle/body weight ratio of WT, Tg-4140, mdx, and mdx;Tg-4140 mice at 10–14 weeks of age (n = 5–10). (C–J) Relative mRNA levels of PGC-1α, utrophin, acetylcholine receptor (AchR) subunits α, δ, ε, and Musk, Erbb1, and Raspn in gastrocnemius muscle of WT, Tg-4140, mdx, and mdx;Tg-4140 mice (n = 3–5). Data are expressed as mean +/− s.e.m. *p<0.05, ** p<0.01, ***p<0.001 by two-tailed unpaired Student's t test.
Figure 7
Figure 7. SIRT1 overexpression in skeletal muscle alleviates the muscular dystrophic phenotype of mdx mouse.
(A) Serum creatine kinase activity in WT, Tg-4140, mdx, mdx;Tg-4140 mice (8–10 weeks old, n = 10). (B) Representative H&E staining of gastrocnemius muscle from mdx and mdx;Tg-4140 mice and quantification of damaged area (10 sections/genotype, n = 3). (C) Representative Evans Blue staining of gastrocnemius muscle from mdx and mdx;Tg-4140 mice and quantification of staining area (10 sections/genotype, n = 3). (D) Distance run in treadmill exercise by WT and Tg-4140 mice, and mdx and mdx;Tg-4140 mice (12–14 weeks old, n = 10–12). (E) Relative mRNA levels of SIRT1 in gastrocnemius muscle of WT and mdx mice (10–12 weeks old, n = 4). Western blot analysis of protein extracts prepared from gastrocnemius muscle of WT and mdx mice. (F–I) Relative mRNA levels of NAMPT, NMNAT1, NMNAT2, and NMNAT3 in gastrocnemius muscle from WT, Tg-4140, mdx and mdx;Tg-4140 mice (n = 3–5). (J) NAD+ levels in gastrocnemius muscle from WT, Tg-4140, mdx and mdx;Tg-4140 mice (n = 3–5). Data are expressed as mean +/− s.e.m. *p<0.05, **p<0.01, ***p<0.001 by two-tailed unpaired Student's t test.
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