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Indoxyl sulfate inhibits muscle cell differentiation via Myf6/MRF4 and MYH2 downregulation
Stanislas Bataille1,2, Nathalie McKay1, Laetitia Koppe3,4
1Aix Marseille University, INSERM, INRAE, C2VN, Marseille, France.
Background:
Chronic kidney disease (CKD) is associated with a significant decrease in muscle strength and mass, possibly related to muscle cell damage by uremic toxins. Here, we studied in vitro and in vivo the effect of indoxyl sulfate (IS), an indolic uremic toxin, on myoblast proliferation, differentiation and expression of myogenic regulatory factors (MRF)-myoblast determination protein 1 (MyoD1), myogenin (Myog), Myogenic Factor 5 (Myf5) and myogenic regulatory factor 4 (Myf6/MRF4)-and expression of myosin heavy chain, Myh2.
Methods:
C2C12 myoblasts were cultured in vitro and differentiated in myotubes for 7 days in the presence of IS at a uremic concentration of 200 µM. Myocytes morphology and differentiation was analyzed after hematoxylin-eosin staining. MRF genes' expression was studied using reverse transcription polymerase chain reaction in myocytes and 5/6th nephrectomized mice muscle. Myf6/MRF4 protein expression was studied using enzyme-linked immunosorbent assay; MYH2 protein expression was studied using western blotting. The role of Aryl Hydrocarbon Receptor (AHR)-the cell receptor of IS-was studied by adding an AHR inhibitor into the cell culture milieu.
Results:
In the presence of IS, the myotubes obtained were narrower and had fewer nuclei than control myotubes. The presence of IS during differentiation did not modify the gene expression of the MRFs Myf5, MyoD1 and Myog, but induced a decrease in expression of Myf6/MRF4 and MYH2 at the mRNA and the protein level. AHR inhibition by CH223191 did not reverse the decrease in Myf6/MRF4 mRNA expression induced by IS, which rules out the implication of the ARH genomic pathway. In 5/6th nephrectomized mice, the Myf6/MRF4 gene was down-regulated in striated muscles.
Conclusion:
In conclusion, IS inhibits Myf6/MRF4 and MYH2 expression during differentiation of muscle cells, which could lead to a defect in myotube structure. Through these new mechanisms, IS could participate in muscle atrophy observed in CKD.
Insights
Indoxyl sulfate (IS), a uremic toxin, impairs muscle cell differentiation by reducing Myf6/MRF4 and MYH2 expression, contributing to muscle atrophy in chronic kidney disease (CKD). This study reveals a novel mechanism of muscle wasting in CKD patients.
Area of Science:
- Muscle Biology
- Nephrology
- Toxicology
Background:
- Chronic kidney disease (CKD) is linked to reduced muscle strength and mass, potentially due to uremic toxin-induced muscle cell damage.
- Indoxyl sulfate (IS) is a key indolic uremic toxin implicated in CKD-related complications.
Purpose of the Study:
- To investigate the in vitro and in vivo effects of indoxyl sulfate (IS) on myoblast proliferation, differentiation, and the expression of myogenic regulatory factors (MRFs).
- To elucidate the role of the Aryl Hydrocarbon Receptor (AHR) in IS-mediated effects on muscle cells.
Main Methods:
- C2C12 myoblasts were differentiated in the presence of IS (200 µM).
- Gene and protein expression of MRFs (MyoD1, Myog, Myf5, Myf6/MRF4) and MYH2 were analyzed.
- AHR inhibition was employed to assess its role.
- Studies were conducted in 5/6th nephrectomized mice models.
Main Results:
- IS exposure resulted in narrower myotubes with fewer nuclei.
- IS significantly decreased the mRNA and protein expression of Myf6/MRF4 and MYH2.
- AHR inhibition did not reverse the IS-induced decrease in Myf6/MRF4 expression, suggesting the ARH genomic pathway is not involved.
- Myf6/MRF4 was downregulated in the muscles of nephrectomized mice.
Conclusions:
- Indoxyl sulfate inhibits Myf6/MRF4 and MYH2 expression during muscle cell differentiation, potentially impairing myotube structure.
- These findings suggest a novel mechanism by which IS contributes to muscle atrophy in CKD.
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