The initial Pubmed-Medline search yielded 371 studies published before November 2020. We identified another three studies through other sources. After reading titles and abstracts, we excluded 254 manuscripts and 113 articles remained. We then read the full text of these articles and excluded more articles based on our inclusion and exclusion criteria. In the end, 24 publications were analysed quantitatively and compiled in a table. The PRISMA flowchart in worksheet S1 in Supplementary Table 1 summarises the search and selection of publications.
Genes whose gain or loss-of-function significantly changes muscle fibre distribution in mice
Overall we identified 25 genes whose gain or loss-of-function significantly changed the percentages of type 1, 2A, 2X or 2B muscle fibres or myosin heavy chain abundance in at least one muscle in mice. Genes whose gain or loss-of-function significantly changed the percentage of type 1, 2A or 2X fibres are presented in Fig. 1 and genes that affect myosin heavy chain isoform expression in Fig. 2.
Specifically, we identified 13 genes whose loss-of-function (Bdkrb2, Bdnf, Camk4, Ccnd3, Cpt1a, Foxj3, Mapk12, Mstn, Myod1, Nfatc1, Nol3, Thra, Thrb) and 2 genes whose gain-of-function (Foxo1, Ppargc1a) significantly changed the proportions of at least one muscle fibre type. Additionally, we identified one gene whose knock out (Ncor1) and 5 genes whose overexpression (Esrrg, Il15, Ppargc1b, Sirt3, Trib3) significantly altered the expression of at least one myosin heavy chain isoform in mice. Moreover, loss-of-function of two genes (Epas1 and Vgll2) and the gain-of-function of two other genes (Akirin-1 and Sirt1) significantly altered both the proportions of at least one muscle fibre type and significantly changed the expression of at least one myosin heavy chain isoform. The effect sizes of the aforementioned gene manipulations range from a reduction of a fibre type by 37% (Ppargc1a, Plantaris type 2B) to a gain of 28% of a single fibre type (Epas1, soleus type 2B). Genes whose knockout affected more than one fibre type in soleus are Bdkrb2, Camk4, Mpak12, Nol3, Thra and Thrb. The muscle fibre genes Bdnf, Ccnd3, and Mstn affected muscle fibre distribution in thetibialis anterior, Bdnf in the extensor digitorum longus and Nol3 in the plantaris. Related to genes whose overexpression changes muscle fibre types, Foxo1 affected more than one fibre type in the soleus, and Ppargc1a in the plantaris. Associated with myosin heavy chain expression we have Ncor1 knockout affecting the myosin heavy chain expression in the gastrocnemius and quadriceps. On the other hand, overexpression of Esrrg, Il15, Ppargc1b and Sirt3 changed muscle fibre proportions in the gastrocnemius, Il15 and Trib3 in the soleus, Il15 and Ppargc1b in the extensor digitorum longus, and Ppargc1b and Trib3 in the tibialis anterior.
Next, we used the list of 25 muscle fibre genes to answer direct research questions through further bioinformatical analyses.
1) Do muscle fibre proteins interact and do muscle fibre genes share common functional features?
To answer this question we completed a String protein interaction analysis [35] and a Toppgene enrichment analysis [34]. The string analysis suggests interactions in-between muscle fibre genes. Clusters of muscle fibre genes included a cluster of genes that encoded thyroid (the expression of all MYH genes responds to thyroid hormone [36]) and oestrogen hormone receptors (Thra, Thrb, Essrg), a cluster with the transcriptional co-factors and transcription factors Ppargc1a, Ppargc1b, Vgll2, Foxo1, Myod1, Nfatc1, the sirtuins Sirt1 and Sirt3, and a cluster of the circulating factors Mstn and Bdnf as well as the kinases Mapk12 and Camk4.
We also used a ToppGene functional enrichment analysis to identify common features and functions among the muscle fibre genes identified. Specifically, we found 15 muscle fibre genes that regulate gene transcription (Foxo1, Ppargc1a, Ncor1, Ppargc1b, Thra, Thrb, Akirin1, Trib3, Nfatc1, Foxj3, Vgll2, Myod1, Epas1, Sirt1, Esrrg), 5 genes that regulate muscle adaptation to contractile activity (see also below), loading conditions, substrate supply, and environmental factors. Of the 25 muscle fibre genes, 13 genes regulate cellular responses to hormones (Ccnd3, Foxo1, Ppargc1a, Ncor1, Ppargc1b, Thra, Thrb, Mstn, Myod1, Bdnf, Sirt1, Esrrg) and six genes are linked to energy metabolism e.g. in the form of mitochondrial biogenesis (Foxo1, Ppargc1a, Ppargc1b, Camk4, Sirt3, Sirt1) (worksheet S4 in Supplementary Table 1).
2) In what human tissues are muscle fibre genes expressed?
To find out whether muscle fibre genes are primarily expressed in skeletal muscle or elsewhere, we retrieved human gene expression data from the Genotype-Tissue Expression (GTEx) Project website (https://gtexportal.org/home [37]). This analysis reveals that two of the muscle fibre genes, Vgll2 and MyoD1, are exclusively expressed in skeletal muscle. Moreover, Mapk12, Foxo1, and Nol3 are most expressed in human skeletal muscle but also elsewhere.
3) Are muscle fibre genes regulated in response to exercise or inactivity?
To systematically investigate whether muscle fibre genes are regulated by exercise, we retrieved from the MetaMEx website (https://www.metamex.eu/) meta-analysed human muscle fibre gene expression data comparing pre and post exercise or inactivity (Fig. 5. Worksheet S7 in Supplementary Table 1 [38]). To find out whether muscle fibre proteins are phosphorylated and become phosphorylated or dephosphorylated after a bout of human exercise in muscle, we also retrieved published phosphoproteomics data [39]. The gene expression analysis identifies PPARGC1A (which encodes Pgc-1α) and VGLL2 as genes that roughly double their expression after acute bouts of endurance or resistance exercise in human vastus lateralis muscle and that decrease their expression in inactive muscles. EPAS1, which encodes a hypoxia-induced transcription factor also increases its expression after a bout of endurance and resistance exercise, but decreases in response to inactivity. Conversely, MSTN expression decreases after a bout of endurance and resistance exercise but increases in response to inactivity. The expression changes of all muscle fibre genes in response to acute endurance exercise, acute resistance exercise and inactivity are shown in Fig. 5.
This reveals that muscle fibre genes such as PPARGC1A and VGLL2 increase their expression whereas MSTN decreases its expression especially after a bout of endurance exercise. The opposite is true for inactivity.
When analysing muscle protein phosphorylation, we found that Vgll2 Ser261 phosphorylation increased by 30% after maximal muscle contractions in mice (p = 0.07) [40]. In addition, FOXO1, MAPK12, NOL3, NCOR1 and SIRT1 were detected as phosphorylated proteins in human muscle after a single high-intensity exercise bout. However, of these muscle fibre proteins, only MAPK12 Ser362 phosphorylation increased by more than 1.5-fold (p < 0.05) [39]. Collectively this suggests that several muscle fibre genes are regulated in response to acute endurance or resistance exercise or inactivity.
5) What is known about sequence variability of the muscle fibre genes in human exome?
Human fibre type distribution in muscles vary in the human population and this is ≈45% explained by genetics [21]. To determine the frequency of human DNA sequence variants of muscle fibre genes, we retrieved exome sequence data for 60,706 humans [41]. The analysis of this data revealed that each muscle fibre gene had on average 160 missense, 3 loss-of-function and 87 synonymous DNA variants. For BDKRB2, CCND3, NOL3, THRA, NCOR1 and EPAS2 homozygous loss-of-function DNA variants are reported [41]. Together this suggests that exome sequence variability of human muscle fibre genes could at least partially explain the currently poorly explained variability of muscle fibre genes.