Lactate Promotes Myoblast Differentiation and Myotube Hypertrophy via a Pathway Involving MyoD In Vitro and Enhances Muscle Regeneration In Vivo

Abstract

Lactate is a metabolic substrate mainly produced in muscles, especially during exercise. Recently, it was reported that lactate affects myoblast differentiation; however, the obtained results are inconsistent and the in vivo effect of lactate remains unclear. Our study thus aimed to evaluate the effects of lactate on myogenic differentiation and its underlying mechanism. The differentiation of C2C12 murine myogenic cells was accelerated in the presence of lactate and, consequently, myotube hypertrophy was achieved. Gene expression analysis of myogenic regulatory factors showed significantly increased myogenic determination protein (MyoD) gene expression in lactate-treated cells compared with that in untreated ones. Moreover, lactate enhanced gene and protein expression of myosin heavy chain (MHC). In particular, lactate increased gene expression of specific MHC isotypes, MHCIIb and IId/x, in a dose-dependent manner. Using a reporter assay, we showed that lactate increased promoter activity of the MHCIIb gene and that a MyoD binding site in the promoter region was necessary for the lactate-induced increase in activity. Finally, peritoneal injection of lactate in mice resulted in enhanced regeneration and fiber hypertrophy in glycerol-induced regenerating muscles. In conclusion, physiologically high lactate concentrations modulated muscle differentiation by regulating MyoD-associated networks, thereby enhancing MHC expression and myotube hypertrophy in vitro and, potentially, in vivo.

Keywords: MyoD; fiber hypertrophy; lactate; muscle differentiation; myosin heavy chain.

Figure 1

Lactate promoted myogenic differentiation of myoblasts. Cells were differentiated in control medium (Con) or medium containing 10 mM sodium lactate (Lac) for 5 d. (A) Microscopic images of C2C12 myoblasts after the induction of differentiation for the indicated days. Scale bar, 200 μm. (B,C) Differentiated C2C12 cells at day 5 (B) and primary myoblasts at day 3 (C) were stained with an MF-20 anti-myosin heavy chain (MHC) antibody (green) and 4′,6-diamidino-2-phenylindole (DAPI) (blue). Fusion index (%), defined as the percentage of nuclei located within MHC-positive myotubes divided by total nuclei, was calculated as described in Methods. n = 3–4 per group. Results are expressed as mean ± SE. * p < 0.05, ** p < 0.01 vs. Con.

Figure 2

Lactate enhanced expression of myogenic determination protein (Myod) and Myh4 genes and MHC protein in C2C12 myotubes. C2C12 cells (A to D) and primary myoblasts (E) were differentiated in control medium (open bar), or medium containing 10 mM sodium lactate (closed bar) or 10 mM sodium chloride (hatched bar). Cells were harvested on the indicated day of differentiation and messenger RNA (mRNA) expression levels of representative myogenic markers were determined by real-time PCR. (A) Myf5, Myod, and Myog; (B) Myh4; and (C) Myod and Myh4. RNA levels were quantified and normalized to that for GAPDH. Values are each expressed as the fold change compared with day 0 in (A), with control on day 3 in (B), and with control on day 5 in (C), with each the value used for normalization arbitrarily set to 1. (D) Cells were harvested on day 5. MHC protein levels were determined and normalized to those of β-actin. (E) Primary myoblasts were differentiated for 3 d and mRNA expression levels of Myod, Myog, and Myh4 were determined. n = 3–4 per group. Results are expressed as means ± SE. * p < 0.05; ** p < 0.01; versus each control group.

Figure 3

Lactate did not affect the protein synthesis pathway in C2C12 myotubes. (A) C2C12 cells were differentiated in control medium (Con) or medium containing 10 mM sodium lactate (Lac) for 5 d. Cells were harvested on day 5 and protein levels of P70S6K, p-P70S6K, and β-actin were analyzed by immunoblotting. (B) P70S6K phosphorylation levels (p-P70S6K/P70S6K) are expressed as fold change compared with that in the Con group (n = 4–5 per group). Results are expressed as mean ± SE.

Figure 4

Lactate increased mRNA expression of predominantly the fast MHC isoform in C2C12 myotubes. (A) Cells were differentiated in control medium (open bar) or medium containing 10 mM sodium lactate (closed bar) for 5 d. Gene expression levels of Myh7, Myh2, Myh1, and Myh4, encoding MHC subtypes I, IIa, IIx, and IIb, respectively, were determined. Values are expressed as the fold changes, compared with each control (n = 4 per group). ** p < 0.01 versus the control. (B) Differentiation was induced in medium containing the indicated concentration of lactate. Cells were harvested on day 5 and expression levels of Myh1 and Myh4 were determined. Values are expressed as fold changes compared with the level in the 0 mM group (n = 4 per group). Results are expressed as mean ± SE. * p < 0.05, ** p < 0.01, *** p < 0.001, versus the 0 mM group.

Figure 5

Lactate-induced increases in Myh4 promoter activity required specific E-box sequences. (A) Undifferentiated (Dif−) or differentiated (Dif+) C2C12 cells were cotransfected with a reporter vector containing 1.4 Kb of the Myh4 promoter region and a MyoD expression vector (pcDNA–MyoD; black bar) or its control (pcDNA3; white bar). (B) Differentiated C2C12 cells were transfected with a reporter vector containing the 1.4 Kb promoter region. At 24 h after transfection, the cells were incubated in the presence (Lac) or absence (Con) of 10 mM lactate for an additional 48 h. (C) Cells were then transfected with each deletion construct and then incubated in the presence (Lac+; black bar) or absence (Lac−; white bar) of 10 mM lactate for an additional 48 h. Luciferase activity was measured and is expressed as the fold induction corrected for transfection efficiency, based on Renilla luciferase activity. Values are expressed as the fold change compared with the vehicle control, which was arbitrarily set to 1. n = 4 per group. Results are expressed as mean ± SE. Δ p < 0.1, * p < 0.05, ** p < 0.01, § p < 0.05, versus the reporter vector containing 1.4 Kb of the promoter region

Figure 6

Blood lactate levels after intraperitoneal injection of lactate. Mice (eight weeks of age, n = 3) were intraperitoneally injected with sodium lactate (500 mg/kg). Blood lactate levels were measured with a blood lactate analyzer (Lactate Pro2; ARKRAY, Kyoto, Japan) at 0 min, 5 min, 15 min, 1 h, 4 h, and 24 h after injection. Results are expressed as mean ± SE.

Figure 7

Lactate accelerated the regeneration of injured muscle. (A) Experimental scheme for the effects of lactate on damaged muscles induced by glycerol. (B) Representative images of hematoxylin and eosin-stained sections of tibialis anterior (TA) muscles from mice treated with lactate (Lac) or saline control (Con) at 7, 14, and 28 d after glycerol injection (scale bar = 100 µm). (C) Messenger RNA levels of representative myogenic markers were determined by real-time PCR in the harvested muscles at day 7. Results are expressed as mean ± SE. (D) Individual regenerated fiber areas were measured at day 14 and 28 in muscles from control (Con) and lactate-treated (Lac) mice (n = 3 per group). Values are presented in a box-and-whisker plot (boxes are constructed with the intervals between the first and third quartiles of the data distribution; lines in the boxes are medians; positive and negative bars are maximum and minimum individual values in each group, respectively. * p < 0.05, *** p < 0.001. (E) The distributions of fiber areas measured at day 28 muscles from control (Con; closed circle) and lactate-treated (Lac; open square) mice are shown.