Abstract

   Embryonic muscle fiber formation determines post‐birth muscle fiber
   totals. The previous research shows SYISL knockout significantly
   increases muscle fiber numbers and mass in mice, but the mechanism
   remains unclear. This study confirms that the SYISL gene, maternal gut
   microbiota, and their interaction significantly affect the number of
   muscle fibers in mouse embryos through distinct mechanisms, as SYISL
   knockout alters maternal gut microbiota composition and boosts butyrate
   levels in embryonic serum. Both fecal microbiota transplantation and
   butyrate feeding significantly increase muscle fiber numbers in
   offspring, with butyrate inhibiting histone deacetylases and increasing
   histone acetylation in embryonic muscle. Combined analysis of RNA‐seq
   between wild‐type and SYISL knockout mice with ChIP‐seq for H3K9ac and
   H3K27ac reveals that SYISL and maternal microbiota interaction
   regulates myogenesis via the butyrate‐HDAC‐H3K9ac/H3K27ac pathway.
   Furthermore, scRNA‐seq analysis shows that SYISL knockout alone
   significantly increases the number and proportion of myogenic cells and
   their dynamics, independently of regulating histone acetylation levels.
   Cell communication analysis suggests that this may be due to the
   downregulation of signaling pathways such as MSTN and TGFβ. Overall,
   multiple pathways are highlighted through which SYISL influences
   embryonic muscle development, offering valuable insights for treating
   muscle diseases and improving livestock production.

   Keywords: butyrate, gut microbiota, muscle fiber number, scRNA‐seq,
   SYISL
     __________________________________________________________________

   SYISL knockout significantly promotes embryonic muscle development and
   increases muscle fiber numbers of offspring by modulating maternal gut
   microbiota composition and butyrate‐HDAC‐H3K9ac/H3K27ac pathway.
   Furthermore, scRNA‐seq analysis shows that SYISL knockout alone
   significantly increases the number and proportion of myogenic cells and
   their dynamics, which may be due to the downregulation of signaling
   pathways such as MSTN and TGFβ.

   graphic file with name ADVS-12-2410953-g004.jpg

1. Introduction

   Understanding the formation of muscle fibers is integral for developing
   methods of treating various muscle diseases for the improvement of meat
   production. The growth and development of skeletal muscle involve two
   stages: the formation of embryonic muscle fibers and the enlargement of
   muscle fibers after birth, in which the number of muscle fibers during
   the embryonic period determines the quantity of muscle fibers after
   birth.^[ [56]^1 ^] In the process of mouse embryonic development,
   primary muscle fibers, which predominantly express the slow myosin
   heavy chain (MyHC I) protein, are formed between embryonic days 11.5
   and 14.5 (E11.5d–E14.5d). Subsequently, secondary muscle fibers are
   formed until E18.5d, and thereafter the number of muscle fibers remains
   stable.^[ [57]^2 ^] The development of embryonic muscle fibers is
   influenced by multiple factors such as transcription factors,^[ [58]^3
   ^] epigenetic modifiers,^[ [59]^4 ^] and non‐coding RNAs.^[ [60]^5 ^]
   The absence of the transcription factor KLF4 results in a significant
   reduction in both the weight of embryos at E17.5d and the area of
   muscle fibers.^[ [61]^6 ^] Knockout (KO) of KDM4A, a histone
   demethylase, inhibits myogenic differentiation by reducing the
   demethylation of the histone marker H3K9me3 at the regulatory regions
   of key myogenic genes such as MyoD and MyoG, leading to reduced
   embryonic weight and decreased muscle fiber size in mice.^[ [62]^7 ^]

   Recent studies have established key interaction relationship between
   host genes and gut microbes,^[ [63]^8 ^] revealing host genes can
   influence the composition of gut microbiota or their metabolic
   products,^[ [64]^9 ^] subsequently affecting muscle development.^[
   [65]^10 ^] For example, MSTN deficiency leads to the enrichment of
   microbes in the colon that produce short‐chain fatty acids (SCFAs),
   thus increasing valeric acid production, which activates the Akt/mTOR
   pathway via G protein‐coupled receptor 43 (GPR43), eventually
   stimulating the growth of type IIb muscle fibers.^[ [66]^11 ^]
   Transplanting the microbiota of Rongchang pigs into germ‐free (GF) mice
   has been shown to alter mice muscle composition, resulting in an
   increase of the type I muscle fiber proportion and a reduction in the
   measured area of type IIb muscle fibers.^[ [67]^12 ^] SCFAs are the
   most extensively studied microbial metabolites in the gut‐muscle axis,
   with numerous SCFAs receptors such as GPR41 and GPR43 present in muscle
   tissue.^[ [68]^13 ^] SCFAs can affect skeletal muscle development by
   activating multiple signaling pathways, such as AMP‐activated protein
   kinase (AMPK), peroxisome proliferator‐activated receptor δ (PPAR‐δ),
   and peroxisome proliferator‐activated receptor‐γ coactivator‐1α
   (PGC‐1α), or by inhibiting histone deacetylases (HDACs) activity.^[
   [69]^14 ^] SCFAs are small molecular substances, that can be
   transferred from the mother through the placenta to the fetus,^[
   [70]^15 ^] while sources have also shown that adding sodium butyrate to
   pregnant rats can significantly reduce blood pressure and levels of
   inflammatory factors, and increase the weight of the fetus and
   placenta.^[ [71]^16 ^]

   Our previous studies identified Synaptopodin‐2 intron sense‐overlapping
   lncRNA (SYISL) as a novel suppressor of muscle growth in mice, pigs,
   and humans,^[ [72]^17 ^] and demonstrated that knockout of SYISL could
   result in a significant increase in the total number of muscle fibers
   and muscle mass in mice.^[ [73]^18 ^] However, how SYISL influences
   offspring embryonic myogenesis is largely unknown. In this study, we
   discovered that the interaction between maternal SYISL and gut microbes
   significantly increases the number of embryonic muscle fibers.
   Specifically, SYISL knockout significantly altered the composition of
   the maternal intestinal microbiota, leading to a significant increase
   in the abundance of Prevotella and butyrate‐producing bacteria, which
   in turn significantly raised the levels of butyrate in the maternal
   serum, feces, and fetal serum. Concurrently, we also discovered that
   butyrate was involved in the significant increase in the number of
   embryonic muscle fibers caused by the SYISL knockout through the
   HDAC‐H3K27ac/H3K9ac pathway. Moreover, single‐cell RNA sequencing
   (scRNA‐seq) of WT and KO fetal muscles from the same heterozygous
   parents showed that SYISL knockout leads to a significant increase in
   myogenic cells and the number of embryonic muscle fibers, likely
   through various key signaling pathways such as TGFβ, MSTN, and PTN. Our
   study reveals the multiple pathways by which SYISL regulates embryonic
   myogenesis in offspring.

2. Results

2.1. Interaction Between Maternal SYISL and Gut Microbes Significantly
Affects Numbers of Secondary and Total Muscle Fibers in Offspring

   To determine interaction between host SYISL gene and gut microbiota and
   its potential effects on muscle fiber development in offspring, we
   prepared germ‐free WT and KO mice (ABT‐WT and ABT‐KO) and established
   four mating groups (Figures [74]1A, [75]S1A, Supporting Information).
   We performed immunofluorescence staining for laminin and MyHC I in the
   leg muscles of E18.5d embryos, and the number of muscle fibers in the
   extensor digitorum longus (EDL) muscles were counted (Figure [76]1B).
   The results showed a significant increase in the number of primary,
   secondary, and total muscle fibers in KO fetuses compared to WT fetuses
   (p < 0.05), along with a significant decrease in the muscle fiber area
   in KO fetuses (Table [77]1 and Figure [78]1C). KO fetuses showed higher
   protein expression levels of the differentiation marker MyoG and MyHC,
   but lower protein expression levels of the fusion marker MYMK in leg
   muscles than WT fetuses (Figure [79]1D). These results were consistent
   with those at postnatal day 0.5 (Figure [80]S1B–E, Supporting
   Information). Additionally, DAPI staining of isolated individual muscle
   fibers showed that the number of nuclei per fiber in KO mice was
   significantly lower than that in WT mice (p < 0.01, Figure [81]1E).

Figure 1.

   Figure 1
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   The interaction between host genes and gut microbes in female mice has
   significant effects on the number of muscle fibers in their offspring.
   A) After antibiotic treatment, female mice of either SPF or
   pseudo‐germ‐free were mated with male mice with the corresponding
   microbial status. Samples were collected on gestational day 18.5. n =
   9. B) Representative images of immunofluorescence staining for laminin
   (red) and MyHC I (green) in the cross‐sections of the hind limbs of KO
   and WT fetuses at E18.5d. The white dashed lines delineate the mouse
   EDL muscle. Scale bars, 50 µm. n = 5. C) Analysis of EDL muscle fiber
   cross‐sectional areas in E18.5d mice. The left figure presents a
   scatter plot of the areas of each muscle fiber, and the right figure
   shows the distribution of the muscle fibers with different area ranges.
   Compared to WT mice, KO mice exhibit a higher proportion of muscle
   fibers with smaller cross‐sectional areas. In an independent
   experiment, the areas of 100 myofibers from the EDL muscle of each
   mouse were analyzed. Data were presented as mean ± SDs, n = 3. ***p <
   0.001. D,F) Western blotting results showed that in both SPF (D) and
   ABT (F) mice, SYISL KO significantly increased the protein expression
   levels of MyoG and MyHC genes and notably decreased the protein
   expression levels of MYMK genes. Data were presented as mean ± SDs, n =
   3. *p < 0.05, **p < 0.01. E) Representative images of DAPI staining of
   single myofibers and quantification of four independent experiments
   show that SYISL knockout significantly decreases the number of nuclei
   per fiber. Scale bar, 50 µm. Twenty single myofibers/muscle/mouse were
   analyzed in an independent experiment. Data were presented as mean ±
   SDs, **p < 0.01.

Table 1.

   Analysis of muscle fiber number of four different types of fetuses at
   E18.5d.
   Type Primary muscle fibers number Secondary muscle fiber number Total
   muscle fiber number
   WT E18.5d 164.75 ± 25.53^b 605.75 ± 15.57^b 770.5 ± 25.39^c
   KO E18.5d 204.8 ± 11.01^c 799.4 ± 25.41^c 1004.2 ± 33.88^d
   ABT‐WT E18.5d 103.25 ± 7.75^a 446.5 ± 39.05^a 549.75 ± 43.62^a
   ABT‐KO E18.5d 124.5 ± 8.79^a 581.75 ± 22.61^b 706.25 ± 28.10^b
   [83]Open in a new tab

   Values with the same number of letters indicate that the difference is
   not significant. Different letters indicate that the difference is
   significant. The data represent the means ± SDs, n = 5.

   Immunofluorescence results showed that ABT‐WT fetuses exhibited a
   significant reduction in the number of primary, secondary, and total
   muscle fibers at E18.5d in comparison to untreated WT fetuses, and
   similar results were observed between ABT‐KO and untreated KO fetuses
   (p < 0.01, Table [84]1 and Figure [85]1B). Western blotting results
   revealed a significant decrease in the differentiation and fusion
   capabilities of embryonic skeletal muscle after antibiotics treatment
   (Figure [86]S1F,G, Supporting Information). ABT‐KO fetuses also showed
   an increase in muscle fiber numbers compared to ABT‐WT mice
   (Table [87]1). The differences in primary, secondary, and total muscle
   fiber numbers between WT and KO fetuses were more prominent than
   between ABT‐KO and ABT‐WT fetuses (Table [88]1 and Figure [89]1B). The
   differences in MYMK, MyoG, and MyHC protein levels in leg muscles
   between WT and KO fetuses were also larger than those between ABT‐WT
   and ABT‐KO fetuses (Figure [90]1D,F). These results suggest that
   maternal gut microbiota had shown effects on embryonic muscle fiber
   formation. Two‐way analysis of variance (ANOVA) analysis of the above
   data further confirmed that the interaction between the maternal SYISL
   genotype and gut microbiota significantly affected on the numbers of
   secondary and total muscle fibers (p < 0.05), whereas the maternal
   SYISL genotype alone and gut microbiota alone affected the numbers of
   primary, secondary, and total muscle fibers (Table [91]2 ). In summary,
   the SYISL gene, maternal gut microbiota, and their interaction had
   notable effects on the formation of embryonic muscle development.

Table 2.

   Results of two‐way ANOVA.
   Type Source of variance Sum of squares df Mean square F p
   Primary muscle fibers Genotype 4440.2 1 4440.2 18.602 0.001**
   Gut microbe 25063.2 1 25063.2 104.999 <0.001***
   Genotype × Gut microbe 480.2 3 160.1 2.012 0.175
   Secondary muscle fibers Genotype 135136.8 1 135136.8 185.902 <0.001***
   Gut microbe 177472.8 1 177472.8 244.142 <0.001***
   Genotype × Gut microbe 4032.8 3 1344.3 5.548 0.032*
   Total muscle fibers Genotype 188568.2 1 188568.2 166.495 <0.001***
   Gut microbe 335923.2 1 335923.2 296.601 <0.001***
   Genotype × Gut microbe 7296.2 3 2432.1 6.442 0.022*
   [92]Open in a new tab

   Two‐factor ANOVA was used to study the impact of genotype and gut
   microbiota on primary, secondary, and total muscle fibers. The data
   represent the means ± SDs of five independent experiments; *p < 0.05,
   **p < 0.01, ***p < 0.001.

2.2. SYISL Knockout Changes the Gut Microbiota Composition and Metabolites of
Pregnant Mice

   Since RT‐qPCR results showed that SYISL gene expression significantly
   increased from E13.5 to E18.5d (Figure [93]2A), we collected female
   mouse fecal samples at E13.5d, E15.5d, and E18.5d from WT and KO mice
   and analyzed the composition of the fecal microbiota by 16S rRNA
   sequencing. These results showed that at E13.5d, the Chao1 index was
   significantly lower in the KO group in the WT group (p < 0.01). As
   pregnancy progressed, the Chao1 index in the KO group significantly
   increased at E15.5d compared to E13.5d (p < 0.05), and both the WT and
   KO groups exhibited significantly higher Chao1 index (p < 0.01) and
   Shannon index at E18.5d than at E13.5d (p < 0.05, Figure [94]2B; Figure
   [95]S2A, Supporting Information). Principal coordinate analysis (PCoA)
   showed that during all three stages of pregnancy (E13.5, E15.5, and
   E18.5d), the fecal microbiota of the KO group significantly separated
   from that of the WT group (p < 0.05, Figure [96]2C). As pregnancy
   progressed, the microbial structure in the feces of the WT mice
   remained relatively stable (p > 0.05, Figure [97]S2B, Supporting
   Information), whereas that of KO mice exhibited significant differences
   across the three pregnancy stages (p < 0.05, Figure [98]S2C, Supporting
   Information). These results suggesting that SYISL KO might
   substantially alter the composition of the microbiota with a
   significant change occurring at E15.5d (secondary fiber formation
   stage). Our analysis of the bacterial differential abundance at the
   phylum showed that during all three stages of pregnancy, the abundance
   of Tenericutes and TM7 was significantly higher in the KO female mice
   than in WT (p < 0.05, Figure [99]2D–F, Figure [100]S2D, Supporting
   Information), whereas the abundance of Proteobacteria was significantly
   lower in the KO female mice than in WT (Figure [101]S2E, Supporting
   Information). Furthermore, we performed genus‐level analysis
   (Figure [102]2G, Figure [103]S2F, Supporting Information) and found
   that the relative abundance of Prevotella significantly increased (p <
   0.01, Figure [104]2H), whereas that of the genus Ruminococcus
   significantly decreased in the KO group (p < 0.05, Figure [105]2I). We
   validated the accuracy of the 16S rRNA results by RT‐qPCR analysis of
   fecal DNA samples from WT and KO mice collected at E18.5d (Figure
   [106]S3A,B, Supporting Information), and our data showed significant
   differences in the relative abundance of the Prevotella and
   Ruminococcus genera during the critical period of secondary muscle
   fiber development (E15.5d–E18.5d). These findings suggest that two
   genera mentioned might play important roles in muscle fiber
   development.

Figure 2.

   Figure 2
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   SYISL gene knockout in pregnant mice significantly affects the gut
   microbiota composition. A) RT‐qPCR results indicate that the expression
   of the SYISL gene increased with embryonic muscle development. eMyHC
   and MyoD were used as positive references. B) The Chao1 index indicates