Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
, 20 (9), e47892

Succinate Induces Skeletal Muscle Fiber Remodeling via SUNCR1 Signaling

Affiliations

Succinate Induces Skeletal Muscle Fiber Remodeling via SUNCR1 Signaling

Tao Wang et al. EMBO Rep.

Abstract

The conversion of skeletal muscle fiber from fast twitch to slow-twitch is important for sustained and tonic contractile events, maintenance of energy homeostasis, and the alleviation of fatigue. Skeletal muscle remodeling is effectively induced by endurance or aerobic exercise, which also generates several tricarboxylic acid (TCA) cycle intermediates, including succinate. However, whether succinate regulates muscle fiber-type transitions remains unclear. Here, we found that dietary succinate supplementation increased endurance exercise ability, myosin heavy chain I expression, aerobic enzyme activity, oxygen consumption, and mitochondrial biogenesis in mouse skeletal muscle. By contrast, succinate decreased lactate dehydrogenase activity, lactate production, and myosin heavy chain IIb expression. Further, by using pharmacological or genetic loss-of-function models generated by phospholipase Cβ antagonists, SUNCR1 global knockout, or SUNCR1 gastrocnemius-specific knockdown, we found that the effects of succinate on skeletal muscle fiber-type remodeling are mediated by SUNCR1 and its downstream calcium/NFAT signaling pathway. In summary, our results demonstrate succinate induces transition of skeletal muscle fiber via SUNCR1 signaling pathway. These findings suggest the potential beneficial use of succinate-based compounds in both athletic and sedentary populations.

Keywords: SUNCR1; aerobic exercise; fiber type; skeletal muscle; succinate.

Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Figure 1
Figure 1. Effects of succinate on growth performance and serum concentration in mice
Male C57BL/6J mice were fed with chow diet supplemented with 0, 0.5, and 1% SUC for 8 weeks.

(A) Serum SUA level, (B) body weight gain, (C) fat and (D) lean mass and (E) gastrocnemius index.

(F) Gastrointestinal muscle fiber immunofluorescent laminin staining and (G) frequency histogram of fiber cross‐sectional area. Scale bar in (F) represents 100 μm.

Data information: Results are presented as mean ± SEM (n = 6–8). Different letters between bars mean  0.05 in one‐way ANOVA analyses followed by post hoc Tukey's tests. *: significant difference ( 0.05) between 0.5% SUC and control group by non‐paired Student's t‐test. #: significant difference ( 0.05) between 1% SUC and control group by non‐paired Student's t‐test.
Figure EV1
Figure EV1. Effects of succinate on growth performance and muscle fiber composition in mice (related to Figs 1 and 3)
Male C57BL/6J mice were fed with chow diet supplemented with 0, 0.5%, or 1% SUC for 8 weeks.

(A) Cumulative food intake and (B) liver index of mice treated with SUC for 8 weeks (n = 8).

Immunoblots and quantification of p‐mTOR, mTOR, p‐FoxO3a, FoxO3a, p‐AKT, and AKT protein in gastrocnemius (n = 3).

Representative images and quantification of laminin (green), MyHC I (red), and MyHC IIb (red) immunofluorescent staining in the (E, F) soleus and (G, H) extensor digitorum longus muscle. The graphs show the MyHC I and MyHC IIb fiber ratios (n = 6). Scale bar in (E, G) represents 100 μm.

The percentage of SDH positive in the (I, J) gastrocnemius, (K, L) soleus, and (M, N) extensor digitorum longus muscle is shown by SDH enzyme staining. Only darkly stained SDH fibers are treated as SDH‐positive fibers. The graphs show the SDH‐positive fiber ratios (n = 4–6). Scale bar in I, K, and M represents 100 μm.

Data information: Results are presented as mean ± SEM. Different letters between bars mean  0.05 in one‐way ANOVA analyses followed by post hoc Tukey's tests.
Figure 2
Figure 2. Succinate enhances the endurance exercise capacity of skeletal muscle in mice
Male C57BL/6J mice fed with chow diet supplemented with 0, 0.5, and 1% SUC for 8 weeks.

(A) The muscle grip strength, (B) running time in low speed, (C) four‐limb handing time, and (D) running time in high speed.

(E) Serum concentration of RBC and (F) HGB in whole blood.

(G–I) Ex vivo gastrocnemius muscle force, (J) fatigability, (K) glucose consumption, and (L) lactate production were tested.

Data information: Results are presented as mean ± SEM (n = 5–8). Different letters between bars mean  0.05 in one‐way ANOVA analyses followed by post hoc Tukey's tests.
Figure 3
Figure 3. Effects of succinate on MyHC expression in mice
Male C57BL/6J mice were fed with chow diet supplemented with 0, 0.5, and 1% SUC for 8 weeks.

The mRNA expression of MyHC I, MyHC IIa, PGC‐1α, myoglobin, TnnT1 MyHC IIb, MyHC IIx, and TnnT3 in the gastrocnemius muscle (n = 5–6).

Immunoblots and quantification of MyHC I, MyHC IIa, and MyHC IIb protein expression in gastrocnemius (n = 3–4).

Representative images and quantification of laminin (green), MyHC I, and MyHC IIb immunofluorescent staining (red) in gastrocnemius (n = 3). Scale bar in (C) represents 100 μm.

Data information: Results are presented as mean ± SEM. Different letters between bars mean  0.05 in one‐way ANOVA analyses followed by post hoc Tukey's tests.
Figure 4
Figure 4. Succinate promotes skeletal muscle mitochondrial biosynthesis and aerobic oxidation in mice
Male C57BL/6J mice were fed with chow diet supplemented with 0 and 1% SUC for 6 weeks.

The O2 consumption (VO2) (A, B) and respiratory exchange ratio (RER) (C, D).

Serum concentration of (E) NEFA in whole blood. The enzymes activity of (F) SDH, (G) HK, and (H) LDH in gastrocnemius.

Immunoblots and quantification of p‐AMPK, PGC‐1α, and myoglobin in gastrocnemius. The same lysates were used for the detection of PGC1α (100 kDa, Fig 4I), myoglobin (17 kDa, Fig 4I), myosin heavy chain (180 kDa, Fig 3B), and tubulin (48 kDa, shared in both Figs 3B and 4I).

Quantification of mitochondrial and electron transport chain (ETC)‐related gene expression i respiratory exchange n gastrocnemius.

OCRs were measured under basal condition in gastrocnemius.

Data information: Results are presented as mean ± SEM (n = 4–6). Different letters between bars mean  0.05 in one‐way ANOVA analyses followed by post hoc Tukey's tests. * 0.05 and ** 0.01 by non‐paired Student's t‐test.
Figure 5
Figure 5. Effects of succinate on MyHC expression, mitochondria biosynthesis, and metabolism in C2C12 cells
C2C12 cells were treated with 0, 0.5, and 2 mM SUC for 48 h.

Representative images and quantification of MyHC I and MyHC IIb immunofluorescent staining (green) in C2C12 cells (n = 16).

The enzymes activity of (C) SDH, (D) LDH, and (E) lactate production in C2C12 cells.

Quantification of mitochondrial DNA contents in C2C12 cells.

(G) Mitochondrial electron microscopy showed the (H) mitochondrial density, (I) mitochondrial coverage, and (J) average mitochondrial area in C2C12 cell. Scale bar in (A) represents 50 μm; scale bar in (G) represents 0.5 μm.

Data information: Results are presented as mean ± SEM (n = 6–8). Different letters between bars mean  0.05 in one‐way ANOVA analyses followed by post hoc Tukey's tests. * 0.05 by non‐paired Student's t‐test.
Figure EV2
Figure EV2. Effects of succinate on muscle fiber and mitochondrial function of C2C12 cells (related to Fig 5)
C2C12 cells were treated with 0, 0.5 mM, or 2 mM SUC for 48 h.

The mRNA expression of MyHC I, MyHC IIa, PGC‐1α, myoglobin, TnnT1 MyHC IIb, MyHC IIx, and TnnT3 in C2C12 cells.

Fluorescence‐activated cell sorting (FACS) analysis of TMRM fluorescence intensity and the relative mean fluorescence intensity of TMRM.

Data information: Results are presented as mean ± SEM (n = 5–6). Different letters between bars mean  0.05 in one‐way ANOVA analyses followed by post hoc Tukey's tests.
Figure 6
Figure 6. SUNCR1 is required for succinate to induce the fiber‐type transition in myotubes

SUNCR1 protein expression in the gastrocnemius from sedentary or post‐running mice (n = 4).

The mRNA level of SUNCR1 in gastrocnemius and soleus (n = 7–8).

[Ca2+]i in C2C12 cells treated with 0 or 2 mM SUC (n = 18–20).

NFAT protein expression in nucleus and cytoplasm of gastrocnemius 0.5 h or 3 h after i. p. injection of 15 mg/kg succinate in C57BL/6J mice (n = 4).

SUNCR1 protein expression in C2C12 cells transfected with vector or siSUNCR1 (n = 3).

(I) [Ca2+]i, and enzymes activity (n = 9–10)of (J) HK, (K) LDH, and (L) SDH in vector or siSUNCR1 transfected C2C12 cells treated with 0 or 2 mM SUC (n = 5–6).

Representative images and quantification of MyHC I and MyHC IIb immunofluorescent staining (green) in C2C12 cells (n = 3). Scale bar in (M) represents 50 μm.

Data information: Results are presented as mean ± SEM. * 0.05 by non‐paired Student's t‐test.
Figure EV3
Figure EV3. Role of SUNCR1/PLC‐β in succinate‐induced in vitro fiber‐type transition in myotubes (related to Fig 6)

[Ca2+]i of C2C12 cells treated with vehicle, SUC (2 mM), SUC (2 mM) + PLC‐β inhibitor U73122 (1 μM), or SUC (2 mM) + PLC‐β inhibitor U73122 (10 μM; n = 9–10).

After 6 days of differentiation, C2C12 cells were treated with vehicle, SUC (2 mM), U73122 (5 μM), or SUC (2 mM) + U73122 (5 μM) for 48 hrs. Representative images (C) and (D, E) quantification of MyHC I and MyHC IIb immunofluorescent staining (green) in the C2C12 cells (n = 3). Scale bar in (C) represents 50 μm.

C2C12 cells were transfected with vector or siSUNCR1, cultured for 6 days in a differentiation medium, and then treated with SUC (2 mM) for 48 h to test the concentration of lactic acid in medium (n = 5–6).

Data information: Results are presented as mean ± SEM. * 0.05 by non‐paired Student's t‐test.
Figure EV4
Figure EV4. SUNCR1 global knockout blocks the effect of succinate on protein synthesis (related to Fig 7)

Schematic representation of SUNCR1 KO by Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) strategy. The sgRNA sites were located in intron 1 and intron 2 of SUNCR1 gene. Four sgRNAs were designed to delete exon 2 of SUNCR1 gene. The DNA sequences contained sgRNA‐binding regions are labeled with lines.

Immunoblots of SUNCR1 protein in liver, fat, soleus (sol), and gastrocnemius (gas) from WT and SUNCR1 KO mice.

Representative images for genotyping screen of WT, heterozygous (Het), and homozygous (KO) SUNCR1 KO mice.

(D) Cumulative food intake, (E) lean mass, (F) fat mass, and (G) body weight gain of WT or SUNCR1 KO mice after 6 weeks of dietary supplementation of 0 or 1% SUC.

Immunoblots and quantification of p‐mTOR, mTOR, p‐FoxO3a, FoxO3a, p‐AKT, and AKT proteins in gastrocnemius from WT or SUNCR1 KO mice after 6 weeks of dietary supplementation of 0 or 1% SUC (n = 3).

Data information: Results are presented as mean ± SEM (n = 5–6). * 0.05 by non‐paired Student's t‐test.
Figure 7
Figure 7. SUNCR1 global knockout blocks the effect of succinate on muscle fiber switch in vivo
Male C57BL/6J or SUNCR1 KO mice were fed with chow diet supplemented with 0 or 1% SUC for 6 weeks.

(A, B) The O2 consumption (VO2), (C, D) RER, (E) muscle grip strength, (F) four‐limb handing time, and (G) low‐speed running time.

The enzymes activity of (H) HK, (I) LDH, and (J) SDH in gastrocnemius.

Immunoblots and quantification of MyHC I, MyHC IIb, NFAT, and PGC‐1α protein in gastrocnemius.

Representative images and quantification of laminin (green), or MyHC I and MyHC IIb (red) immunofluorescent staining in gastrocnemius muscle (n = 3). Scale bar in (M) represents 100 μm.

Data information: Results are presented as mean ± SEM (n = 5–6). * 0.05 and ** 0.01 by non‐paired Student's t‐test.
Figure 8
Figure 8. Gastrocnemius‐specific SUNCR1 knockdown abolishes the effect of succinate on muscle fiber switch in vivo
Male C57BL/6J mice were injected with LV‐shScrambled or shSUNCR1 lentivirus specifically into the gastrocnemius at 6 weeks of age. After 2 weeks of recovery, mice were fed with chow diet supplemented with 0 or 1% SUC for 6 weeks.

Timeline of the experimental protocol.

SUNCR1 protein expression in gastrocnemius from mice transfected with shSUNCR1 lentivirus or LV‐shScrambled (n = 3).

(C) The running time in low speed, (D) four‐limb handing time, and (E) muscle grip strength of both control and gastrocnemius‐specific SUNCR1 knockdown mice.

The enzymes activity of (F) HK, (G) LDH, and (H) SDH in gastrocnemius.

Immunoblots and quantification of MyHC I, MyHC IIb, NFAT, and PGC‐1α protein in gastrocnemius (n = 3). Data information: Results are presented as mean ± SEM (n = 5–8). * 0.05 by non‐paired Student's t‐test.

Figure EV5
Figure EV5. Effects of gastrocnemius‐specific SUNCR1 knockdown on body weight (related to Fig 8)

Male C57BL/6J mice were injected with LV‐shScrambled or shSUNCR1 lentivirus specifically into the gastrocnemius at 6 weeks of age. After 2 weeks of recovery, mice were fed with chow diet supplemented with 0 or 1% SUC for 6 weeks. (A) Cumulative food intake, (B) body weight gain, (C) fat mass, and (D) lean mass of mice after 6 weeks of dietary SUC supplementation.

Data information: Results are presented as mean ± SEM (n = 7–8).

Similar articles

See all similar articles

References

    1. Zierath JR, Hawley JA (2004) Skeletal muscle fiber type: influence on contractile and metabolic properties. PLoS Biol 2: e348 - PMC - PubMed
    1. Schiaffino S, Reggiani C (2011) Fiber types in mammalian skeletal muscles. Physiol Rev 91: 1447–1531 - PubMed
    1. Bassel‐Duby R, Olson EN (2006) Signaling pathways in skeletal muscle remodeling. Annu Rev Biochem 75: 19–37 - PubMed
    1. Holloszy JO, Coyle EF (1984) Adaptations of skeletal muscle to endurance exercise and their metabolic consequences. J Appl Physiol 56: 831–838 - PubMed
    1. Westerblad H, Lee JA, Lannergren J, Allen DG (1991) Cellular mechanisms of fatigue in skeletal muscle. Am J Physiol 261: C195–C209 - PubMed

Publication types

LinkOut - more resources

Feedback