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. 2018 Aug 16;19(8):2418.
doi: 10.3390/ijms19082418.

Metronidazole Causes Skeletal Muscle Atrophy and Modulates Muscle Chronometabolism

Ravikumar Manickam  1 , Hui Yun Penny Oh  2   3 , Chek Kun Tan  4 , Eeswari Paramalingam  5 , Walter Wahli  6   7
Affiliations

Metronidazole Causes Skeletal Muscle Atrophy and Modulates Muscle Chronometabolism

Ravikumar Manickam et al. Int J Mol Sci. .

Abstract

Antibiotics lead to increased susceptibility to colonization by pathogenic organisms, with different effects on the host-microbiota relationship. Here, we show that metronidazole treatment of specific pathogen-free (SPF) mice results in a significant increase of the bacterial phylum Proteobacteria in fecal pellets. Furthermore, metronidazole in SPF mice decreases hind limb muscle weight and results in smaller fibers in the tibialis anterior muscle. In the gastrocnemius muscle, metronidazole causes upregulation of Hdac4, myogenin, MuRF1, and atrogin1, which are implicated in skeletal muscle neurogenic atrophy. Metronidazole in SPF mice also upregulates skeletal muscle FoxO3, described as involved in apoptosis and muscle regeneration. Of note, alteration of the gut microbiota results in increased expression of the muscle core clock and effector genes Cry2, Ror-β, and E4BP4. PPARγ and one of its important target genes, adiponectin, are also upregulated by metronidazole. Metronidazole in germ-free (GF) mice increases the expression of other core clock genes, such as Bmal1 and Per2, as well as the metabolic regulators FoxO1 and Pdk4, suggesting a microbiota-independent pharmacologic effect. In conclusion, metronidazole in SPF mice results in skeletal muscle atrophy and changes the expression of genes involved in the muscle peripheral circadian rhythm machinery and metabolic regulation.

Keywords: circadian rhythm; gut dysbiosis; metronidazole; skeletal muscle atrophy.

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Conflict of interest statement

All authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Metronidazole enhances susceptibility to colonization by Proteobacteria and enrichment of Erysipelotrichales. 16s rRNA genes were sequenced from DNA extracted from the fecal pellets of metronidazole (MET)-treated and nontreated control (CNTL) specific pathogen–free (SPF) mice. (a) Percentage representation of the bacterial phyla distribution in fecal pellets of metronidazole-treated and nontreated control SPF mice demonstrating increased susceptibility to colonization by Proteobacteria, especially Parasutterella. N = 3 mice per group. Data are presented as means ± standard error of the mean (SEM). Asterisks indicate statistically significant differences (*, p < 0.05; ns, nonsignificant with student’s t test). (b) Gradient heatmap showing enrichment of the bacterial species Erysipelotrichales in metronidazole-treated SPF mice as compared to nontreated controls. Relative abundance of the bacterial species is color coded from minimum (red) to maximum (green). N = 3 mice per nontreated control (CNTL1–3) and metronidazole-treated (MET1–3) groups.
Figure 2
Figure 2
Metronidazole causes reduced hind limb muscle weight and myofiber surface area. (a) Metronidazole treatment of SPF mice resulted in a marginal, nonsignificant decrease in body weight compared to nontreated controls. N = 5 mice per group. (b) Absolute hind limb muscle weights of tibialis anterior (TA), extensor digitorum longus (EDL), gastrocnemius (GAS), soleus (SOL), and quadriceps (QUAD) muscles of metronidazole-treated SPF mice compared to nontreated controls. N = 5 mice per group. (c) Myofiber numbers according to myofiber surface area in the tibialis anterior in metronidazole-treated and nontreated SPF mice. N = 3 mice per group. A thousand random myofibers per mouse were counted across the mid-belly region of the tibialis anterior muscle cryosections. Data presented as means ± SEM of 3000 myofibers per group (CNTL and MET). Asterisks indicate statistically significant differences (*, p < 0.05; **, p < 0.01; ***, p < 0.001; ns, nonsignificant with student’s t test).
Figure 3
Figure 3
Metronidazole causes changes in the expression of skeletal muscle atrophy genes. Real-time quantitative PCR analysis of Hdac4, myogenin, MuRF1, atrogin1, and FoxO3 expression in gastrocnemius of metronidazole-treated and nontreated (a) SPF and (b) GF mice. N = 5 mice per group. Data presented as means ± SEM. Asterisks indicate statistically significant differences (*, p < 0.05; **, p < 0.01; ***, p < 0.001; ns, nonsignificant with student’s t test).
Figure 4
Figure 4
Metronidazole affects the expression of skeletal muscle metabolism genes. Real-time quantitative PCR analysis of FoxO1 and Pdk4 expression in gastrocnemius of metronidazole-treated and nontreated (a) SPF and (b) GF mice. N = 5 mice per group. Data presented as means ± SEM. Asterisks indicate statistically significant differences (*, p < 0.05; **, p < 0.01; ns, nonsignificant with student’s t test).
Figure 5
Figure 5
Metronidazole alters skeletal muscle core clock and clock effector gene expression. Real-time quantitative PCR analysis of Bmal1, Per2, Cry2, Ror-β, and E4BP4 expression in gastrocnemius of metronidazole-treated and nontreated (a) SPF and (b) GF mice. N = 5 mice per group. Data presented as means ± SEM. Asterisks indicate statistically significant differences (*, p < 0.05; ***, p < 0.001; ns, nonsignificant with student’s t test).
Figure 6
Figure 6
Metronidazole modulates skeletal muscle adiponectin and PPARγ expression. Real-time quantitative PCR analysis of PPARγ and adiponectin expression in gastrocnemius muscle of metronidazole-treated and nontreated (a) SPF (a) and (b) GF mice. N = 5 mice per group. Data presented as means ± SEM. Asterisks indicate statistically significant differences (*, p < 0.05; **, p < 0.01; ns, nonsignificant with student’s t test).
Figure 7
Figure 7
Metronidazole disrupts skeletal muscle RNA epigenetics. Real-time quantitative PCR analysis of RNA m6A methyltransferases Mettl3 and Mettl14 expression in gastrocnemius muscle of metronidazole-treated and nontreated (a) SPF and (b) GF mice. N = 5 mice per group. Data presented as means ± SEM. Asterisks indicate statistically significant differences (*, p < 0.05; ns, nonsignificant with student’s t test).
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