Circular RNA screening identifies circMYLK4 as a regulator of fast/slow myofibers in porcine skeletal muscles

The type of myofiber is related to the quality of meat. The slow oxidized myofiber helps to increase the tenderness and juiciness of muscle. Numerous studies have shown that circRNA plays a key role in skeletal muscle development. However, the role of circRNA in porcine skeletal myofiber types is unclear. In this study, we performed high-throughput RNA sequencing to study the differential expression of circRNA in the longissimus dorsi and the soleus muscle. A total of 40,757 circRNAs were identified, of which 181 were significantly different. Interestingly, some circRNAs were involved in metabolism pathways, AMPK, FoxO, and PI3K-Akt signaling pathways. Besides, we focused on a novel circRNA-circMYLK4. By injecting circMYLK4-AAV into piglets, we found that circMYLK4 significantly increased the mRNA and protein levels of the slow muscle marker genes. In summary, our study laid an essential foundation for further research of circRNA in myofiber type conversion and higher meat quality.

Analysis of potential regulatory LncRNAs and CircRNAs in the oxidative myofiber and glycolytic myofiber of chickens

SART and PMM are mainly composed of oxidative myofibers and glycolytic myofibers, respectively, and myofiber types profoundly influence postnatal muscle growth and meat quality. SART and PMM are composed of lncRNAs and circRNAs that participate in myofiber type regulation. To elucidate the regulatory mechanism of myofiber type, lncRNA and circRNA sequencing was used to systematically compare the transcriptomes of the SART and PMM of Chinese female Qingyuan partridge chickens at their marketing age. The luminance value (L*), redness value (a*), average diameter, cross-sectional area, and density difference between the PMM and SART were significant (p < 0.05). ATPase staining results showed that PMMs were all darkly stained and belonged to the glycolytic type, and the proportion of oxidative myofibers in SART was 81.7%. A total of 5 420 lncRNAs were identified, of which 365 were differentially expressed in the SART compared with the PMM (p < 0.05). The cis-regulatory analysis identified target genes that were enriched for specific GO terms and KEGG pathways (p < 0.05), including striated muscle cell differentiation, regulation of cell proliferation, regulation of muscle cell differentiation, myoblast differentiation, regulation of myoblast differentiation, and MAPK signaling pathway. Pathways and coexpression network analyses suggested that XR_003077811.1, XR_003072304.1, XR_001465942.2, XR_001465741.2, XR_001470487.1, XR_003077673.1 and XR_003074785.1 played important roles in regulating oxidative myofibers by TBX3, QKI, MYBPC1, CALM2, and PPARGC1A expression. A total of 10 487 circRNAs were identified, of which 305 circRNAs were differentially expressed in the SART compared with the PMM (p < 0.05). Functional enrichment analysis showed that differentially expressed circRNAs were involved in host gene expression and were enriched in the AMPK, calcium signaling pathway, FoxO signaling pathway, p53 signaling pathway, and cellular senescence. Novel_circ_004282 and novel_circ_002121 played important roles in regulating oxidative myofibers by PPP3CA and NFATC1 expression. Using lncRNA-miRNA/circRNA-miRNA integrated analysis, we identified many candidate interaction networks that might affect muscle fiber performance. Important lncRNA-miRNA-mRNA networks, such as lncRNA-XR_003074785.1/miR-193-3p/PPARGC1A, regulate oxidative myofibers. This study reveals that lncXR_003077811.1, lncXR_003072304.1, lncXR_001465942.2, lncXR_001465741.2, lncXR_001470487.1, lncXR_003077673.1, XR_003074785.1, novel_circ_004282 and novel_circ_002121 might regulate oxidative myofibers. The lncRNA-XR_003074785.1/miR-193-3p/PPARGC1A pathway might regulate oxidative myofibers. All these findings provide rich resources for further in-depth research on the regulatory mechanism of lncRNAs and circRNAs in myofibers.

The previous stretching to resistance training promotes adaptations on the postsynaptic region in different myofiber types

This investigation revealed the postsynaptic morphological adaptations in static stretching, resistance training, and their association in adult male Wistar rats. We processed the soleus and plantaris muscle for histochemical (muscle hypertrophy) and postsynaptic region imaging techniques. We observed muscle hypertrophy in both groups submitted to resistance training, even though the cross-section area is larger when there is no previous static stretching. The soleus postsynaptic region revealed an increase in compactness and fragmentation index in combined exercise. The resistance training promotes high adaptations in the postsynaptic area of plantaris; moreover, the previous static stretching decreased this area. In conclusion, the neuromuscular system’s components respond according to the myofiber type even though it is the same physical exercise. Besides, static stretching (isolated or combined) plays a crucial role in neuromuscular adaptations.

Abundant Synthesis of Netrin-1 in Satellite Cell-Derived Myoblasts Isolated from EDL Rather Than Soleus Muscle Regulates Fast-Type Myotube Formation

Resident myogenic stem cells (satellite cells) are attracting attention for their novel roles in myofiber type regulation. In the myogenic differentiation phase, satellite cells from soleus muscle (slow fiber-abundant) synthesize and secrete higher levels of semaphorin 3A (Sema3A, a multifunctional modulator) than those derived from extensor digitorum longus (EDL; fast fiber-abundant), suggesting the role of Sema3A in forming slow-twitch myofibers. However, the regulatory mechanisms underlying fast-twitch myotube commitment remain unclear. Herein, we focused on netrin family members (netrin-1, -3, and -4) that compete with Sema3A in neurogenesis and osteogenesis. We examined whether netrins affect fast-twitch myotube generation by evaluating their expression in primary satellite cell cultures. Initially, netrins are upregulated during myogenic differentiation. Next, we compared the expression levels of netrins and their cell membrane receptors between soleus- and EDL-derived satellite cells; only netrin-1 showed higher expression in EDL-derived satellite cells than in soleus-derived satellite cells. We also performed netrin-1 knockdown experiments and additional experiments with recombinant netrin-1 in differentiated satellite cell-derived myoblasts. Netrin-1 knockdown in myoblasts substantially reduced fast-type myosin heavy chain (MyHC) expression; exogenous netrin-1 upregulated fast-type MyHC in satellite cells. Thus, netrin-1 synthesized in EDL-derived satellite cells may promote myofiber type commitment of fast muscles.

Difference in potential DNA methylation impact on gene expression between fast- and slow-type myofibers

Skeletal muscles are comprised of two major types of myofibers, fast and slow. It is hypothesized that once myofiber type is determined, muscle fiber-type specificity is maintained by an epigenetic mechanism, however, this remains poorly understood. To address this, we conducted a comprehensive CpG methylation analysis with a reduced representation of bisulfite sequencing (RRBS). Using GFP-myh7 mouse, we visually distinguished and separately pooled slow-type and myh7-negative fast-type fibers for analyses. A total of 31,967 and 26,274 CpGs were hypermethylated by ≥10% difference in the fast- and slow-type fibers, respectively. Notably, the number of promoter-hypermethylated genes with down-regulated expression in the slow-type fibers was 3.5 times higher than that in the fast-type fibers. Gene bodies of the fast-type-specific myofibrillar genes Actn3, Tnnt3, Tnni2, Tnnc2, and Tpm1 were hypermethylated in the slow-type fibers, whereas those of the slow-type-specific genes Myh7, Tnnt1, and Tpm3 were hypermethylated in the fast-type fibers. Each of the instances of gene hypermethylation was associated with the respective down-regulated expression. In particular, a relationship between CpG methylation sites and the transcription variant distribution of Tpm1 was observed, suggesting a regulation of Tpm1 alternative promoter usage by gene body CpG methylation. An association of hypermethylation with the regulation of gene expression was also observed in Wdr70, and transcription factors Sim2 and Tbx1. These results suggest not only a myofiber type-specific regulation of gene expression and alternative promoter usage by gene body CpG methylation, but also a dominant effect of promoter-hypermethylation on the gene expressions in slow myofibers.

Ezrin regulated myoblast differentiation/fusion and muscle fiber specialization through PKA-NFAT-MyoD/MEF2csignalling pathway

Backgorund:Neuromuscular diseases are a kind of nervous system diseases that have a high disability rate.Ezrin’ role in skeletal muscle has not been identified. This study aims to confirm the effect and mechanism of Ezrin on myoblast differentiation and fusion, myotube size, and myofiber type.Method:By using immunoassaying and western blot analyses, Ezrin, MyHC,MEF2c, MyoG, PKAα/β/γ, PKA reg Iα, PKA reg IIβand NFATc1-c4 were detected in myoblast cells treated with Ad-Ezrin or Ad-shEzrin. Real-time PCR were used to evaluate MyoD, Myf5, MyHC-I , MyHC-IIa/b and MyHC-IIx in myoblast cells. PKA inhibitor H-89 or PKAreg I activator N6-Bz-cAMP were added into medium to confirm their relationship between Ezrin and PKA during myoblast differentiation/fusion. In vitro, Ad-NFATc1/c2 or Ad-shNFATc3/c4 were respectively transfected into C2C12 cells, myoblast differentiation/fusion, myotube size and myofiber type were assessed by using immunostaining of MyHC, MEF2c and MyoG. In vivo, transfection of Ad-Ezrin into gastrocnemius and soleus muscles for 7 days, the numbers of MyHC-1 postivemyofibers were analyzed after immunostaining of MyHC-1.Results: Ezrin expression were time-dependently increased during myoblast differentiation/fusion. Knockdown of Ezrin by shRNA delayed myoblast differentiation and fusion in a time dose-dependent pattern, as shown by immunostaining of MyHC. Conversely, over-expression of Ezrin by adenovirus time- and dosage-dependently promoted myoblastdifferentiation/fusion, and muscle fiber specialization characterized by increased MyHC I and MyHCIIa/b. Forced expression of Ezrin did not alter PKA, and PKAreg II α levels, but altered the levels of PKAreg I α/β, Myf5 and MyoD, and leading to the accumulation of MyoG+/MEF2c+ nuclei. By contrast, Ezrin knockdown significantly decreased the PKA reg I/II ratio and MyoG+/MEF2c+ nuclei. The PKA inhibitor H-89 remarkably abolished the beneficial effect of over-expressingEzrin on the numbers of MyHC+ myotubes and MyoG+/MEF2c nuclei. These opposite changes mediated by knocking down Ezrin were almost eliminated by PKAreg I activator N6-Bz-cAMP. Furthermore, over-expression of NFATc2 or knockdown of NFATc4reversed the inhibitory effect of Ezrin knockdown on myoblast differentiation/fusion, resulting in the recovery of the numbers ofMyoG+/MEF2c+ nucleiin3-nuclei+myotubes. Meanwhile, overexpression of Ezrin specifically induced type I muscle fiber specialization, which was associated with increased levels of NFATc1/c2. Furthermore, in vivo transfection ofAd-Ezrin into gastrocnemius and soleus muscles increased the numbers of MyHC-1 postivemyofibers. By contrast, knockdown of NFATc4resulted in the recovery to normal levels of MyHC-2b in Ezrin-knockdown myoblast cells, attributingtoregainingMyoDand MEF2c expression. Conclusions: Ezrin trigger myoblast differentiation and fusion, myotube size, and alters muscle fiber specialization through PKA-NFAT-MyoD/MEF2C signalling pathway.

Circular RNA screening identifies circMYLK4 as a regulator of fast/slow myofibers in porcine skeletal muscles

Background: The myofiber type is related to the quality of meat; specifically, slow-oxidized myofiber helps to increase the tenderness and juiciness of meat. An increasing number of studies have shown that circRNAs play a key role in skeletal muscle development. However, the key circRNAs that regulate myofiber types and their roles are still poorly understood.Results: A total of 40757 circRNAs were identified from the longissimus dorsi (LD) and the soleus (Sol) muscles, of which 10388 were co-expressed in the two muscles. Further analysis found 181 differentially expressed circRNAs in the LD compared with Sol. Functional enrichment analysis showed that target genes of differentially expressed circRNA-sponge miRNAs were enriched in the AMPK, FoxO and PI3K-Akt signaling pathways. In addition, we focused on a novel circRNA—circMYLK4. CircMYLK4 significantly increased the mRNA and protein levels of slow muscle marker genes and caused the flesh to turn red.Conclusion: Our study laid an essential foundation for further research on circRNAs in myofiber type conversion and the achievement of higher meat quality.

Microutrophin expression in dystrophic mice displays myofiber type differences in therapeutic effects

Gene therapy approaches for DMD using recombinant adeno-associated viral (rAAV) vectors to deliver miniaturized (or micro) dystrophin genes to striated muscles have shown significant progress. However, concerns remain about the potential for immune responses against dystrophin in some patients. Utrophin, a developmental paralogue of dystrophin, may provide a viable treatment option. Here we examine the functional capacity of an rAAV-mediated microutrophin (μUtrn) therapy in the mdx4cv mouse model of DMD. We found that rAAV-μUtrn led to improvement in dystrophic histopathology & mostly restored the architecture of the neuromuscular and myotendinous junctions. Physiological studies of tibialis anterior muscles indicated peak force maintenance, with partial improvement of specific force. A fundamental question for μUtrn therapeutics is not only can it replace critical functions of dystrophin, but whether full-length utrophin impacts the therapeutic efficacy of the smaller, highly expressed μUtrn. As such, we found that μUtrn significantly reduced the spacing of the costameric lattice relative to full-length utrophin. Further, immunostaining suggested the improvement in dystrophic pathophysiology was largely influenced by favored correction of fast 2b fibers. However, unlike μUtrn, μdystrophin (μDys) expression did not show this fiber type preference. Interestingly, μUtrn was better able to protect 2a and 2d fibers in mdx:utrn-/- mice than in mdx4cv mice where the endogenous full-length utrophin was most prevalent. Altogether, these data are consistent with the role of steric hindrance between full-length utrophin & μUtrn within the sarcolemma. Understanding the stoichiometry of this effect may be important for predicting clinical efficacy.

Circular RNA screening identifies circMYLK4 as a regulator of fast/slow myofibers in porcine skeletal muscles

Background: The myofiber type is related to the quality of meat; specifically, slow-oxidized myofiber helps to increase the tenderness and juiciness of meat. An increasing number of studies have shown that circRNAs play a key role in skeletal muscle development. However, the key circRNAs that regulate myofiber types and their roles are still poorly understood.Results: A total of 40757 circRNAs were identified from the longissimus dorsi (LD) and the soleus (Sol) muscles, of which 10388 were co-expressed in the two muscles. Further analysis found 181 differentially expressed circRNAs in the LD compared with Sol. Functional enrichment analysis showed that target genes of differentially expressed circRNA-sponge miRNAs were enriched in the AMPK, FoxO and PI3K-Akt signaling pathways. In addition, we focused on a novel circRNA—circMYLK4. CircMYLK4 significantly increased the mRNA and protein levels of slow muscle marker genes and caused the flesh to turn red.Conclusion: Our study laid an essential foundation for further research on circRNAs in myofiber type conversion and the achievement of higher meat quality.