Satellite Cells Exhibit Decreased Numbers and Impaired Functions on Single Myofibers Isolated from Vitamin B6-Deficient Mice

Abstract

Emerging research in human studies suggests an association among vitamin B6, sarcopenia, and muscle strength. However, very little is known regarding its potential role at the cellular level, especially in muscle satellite cells. Therefore, to determine whether vitamin B6 affects the satellite cells, we isolated single myofibers from muscles of vitamin B6-deficient and vitamin B6-supplemented mice. Subsequently, we subjected them to single myofiber culture and observed the number and function of the satellite cells, which remained in their niche on the myofibers. Prior to culture, the vitamin B6-deficient myofibers exhibited a significantly lower number of quiescent satellite cells, as compared to that in the vitamin B6-supplemented myofibers, thereby suggesting that vitamin B6 deficiency induces a decline in the quiescent satellite cell pool in mouse muscles. After 48 and 72 h of culture, the number of proliferating satellite cells per cluster was similar between the vitamin B6-deficient and -supplemented myofibers, but their numbers decreased significantly after culturing the myofibers in vitamin B6-free medium. After 72 h of culture, the number of self-renewing satellite cells per cluster was significantly lower in the vitamin B6-deficient myofibers, and the vitamin B6-free medium further decreased this number. In conclusion, vitamin B6 deficiency appears to reduce the number of quiescent satellite cells and suppress the proliferation and self-renewal of satellite cells during myogenesis.

Keywords: amino acids; carnosine; proliferation; sarcopenia; satellite cells; self-renewal; single myofibers; skeletal muscle; vitamin B6.

Figure 1

Experimental procedure of single myofiber culture for assessing satellite cell number and function while the cells remain in their niche on single myofibers. EDL, extensor digitorum longus; SC, satellite cells; B6, vitamin B6 (PN HCl). Created with BioRender.com (accessed on 12 December 2021).

Figure 2

Effects of vitamin B6 deficiency on satellite cell numbers at the quiescent state (0 h) and viability after being activated for 24 h. (A) Representative images demonstrating satellite cells stained with Pax7 (arrows) and MyoD on a single fiber at 24 h of culture (scale bar = 50 μm). (B) Quantified data showed the number of quiescent satellite cells on myofibers without culturing (0 h of culture) (4 mice were used per group, ≥15 myofibers per mouse; n = 65 myofibers (35 mg) and n = 77 (1 mg)). (C) Quantified data showed the number of activated satellite cells on myofibers following 24 h of culture (3–4 mice were used per group, ≥15 myofibers per mouse; n = 95 myofibers (35 mg), n = 65 myofibers (1 mg), and n = 50 myofibers (1 mg (−))). (D) Comparison of the number of satellite cells per myofibers between 0 h (b) and 24 h (c) of culture. 1 mg (−) indicates the vitamin B6-deficient myofibers cultured in the vitamin B6-free medium. 35 mg PN HCl/ kg and 1 mg PN HCl/kg represent the vitamin B6-supplemented group and the vitamin B6-deficient group, respectively. Values represent the means ± SD. p value < 0.05 was considered statistically significant (unpaired t-test). ns = no significance.

Figure 3

Effects of vitamin B6 deficiency on satellite cell function (proliferation, self-renewal, and differentiation) following 48 and 72 h of culture. (A) Representative images of single myofibers after 48 or 72 h of culture co-immunostained for Pax7 and MyoD (arrows) (scale bar = 50 μm (T48) or 20 μm (T72)). (B–D) Following 48 h of culture, quantified data showed the number of clusters per myofiber (B), the number of Pax7⁺, Pax7⁺/MyoD⁺, or MyoD⁺ cells per myofiber (C), and the number of the positive cells per cluster recalculated from the (B) and (C) data (D). Four mice were used per group, ≥15 myofibers per mouse; n = 83 myofibers (35 mg), n = 78 (1 mg), and n = 70 myofibers (1 mg (−)). (E–G) Following 72 h of culture, quantified data showed the number of clusters per myofiber (E), the number of Pax7⁺, Pax7⁺/MyoD⁺, or MyoD⁺ cells per myofiber (F), and the number of the positive cells per cluster recalculated from the (e) and (f) data (G). Four mice were used per group, ≥15 myofibers per mouse; n = 65 myofibers (35 mg), n = 65 (1 mg), and n = 74 myofibers (1 mg (−)). 1 mg (−) indicates the vitamin B6-deficient myofibers cultured in the vitamin B6-free medium. 35 mg PN HCl/ kg and 1 mg PN HCl/kg represent the vitamin B6-supplemented group and the vitamin B6-deficient group, respectively. Values represent the means ± SD. p value < 0.05 was considered statistically significant (Unpaired t-test). ns = no significance.

Figure 4

Effects of vitamin B6 deficiency on levels of imidazole dipeptides and their substrate, β-alanine, in skeletal muscles. (A) Levels of β-alanine, (B) levels of carnosine, and (C) levels of anserine. 35 mg PN HCl/ kg and 1 mg PN HCl/kg represent the vitamin B6-supplemented group and the vitamin B6-deficient group, respectively. Values represent the means ± SD (n = 10). p value < 0.05 was considered statistically significant (unpaired t-test).

Figure 5

Effects of vitamin B6 on carnosine biosynthesis in C2C12 muscle cells. (A) Levels of carnosine and (B) levels of β-alanine. B6 (+) and B6 (−) represent the normal medium with vitamin B6 (PN) or the vitamin B6 (PN)-deficient medium, respectively, used in C2C12 cell culture. Values represent the means ± SD (n = 6 wells from a 6-well plate). p value < 0.05 was considered statistically significant (Unpaired t-test).

Figure 6

Events during myogenic progression upon injury and effects of vitamin B6 deficiency on satellite cells. SC, satellite cells; B6, vitamin B6 (PN HCl). Created with BioRender.com (accessed on 12 December 2021).