Itaconate and its derivatives repress C2C12 myogenesis

A Krebs cycle intermediate metabolite, itaconate, has gained attention as a potential antimicrobial and autoimmune disease treatment due to its anti-inflammatory effects. While itaconate and its derivatives pose an attractive therapeutic option for the treatment of inflammatory diseases, the effects outside the immune system still remain limited, particularly in the muscle. Therefore, we endeavored to determine if itaconate signaling impacts muscle differentiation. Utilizing the well-established C2C12 model of in vitro myogenesis, we evaluated the effects of itaconate and its derivatives on transcriptional and protein markers of muscle differentiation as well as mitochondrial function. We found itaconate and the derivatives dimethyl itaconate and 4-octyl itaconate disrupt differentiation media-induced myogenesis. A primary biological effect of itaconate is a succinate dehydrogenase (SDH) inhibitor. We find the SDH inhibitors dimethyl malonate and harzianopyridone phenocopie the anti-myogenic effects of itaconate. Furthermore, we find treatment with exogenous succinate results in blunted myogenesis. Together our data indicate itaconate and its derivatives interfere with in vitro myogenesis, potentially through inhibition of SDH and subsequent succinate accumulation. More importantly, our findings suggest the therapeutic potential of itaconate and its derivatives could be limited due to deleterious effects on myogenesis.

Introduction expression levels although it did not change MYOD levels (Fig. 1f). MYHC was visualized 94 by immunofluorescence at day 4 of differentiation ( Fig. 1g) and myogenic index was 95 significantly impaired by DMI (Fig. 1h) and 4-OI (Fig. 1i).  Heatmap depiction of average fold change (Sig. denotes statistical differences at day 4, n = 3) in myogenic gene transcription levels detected via RT-qPCR when exposed to differential media with or without (a) dimethyl itaconate (DMI: 125 µM) and (b) 4-octyl itaconate (4-OI: 125 µM) relative to the untreated group over a 4-day time course. Fold change of Myog mRNA expression at various time points comparing differentiation media (DM) control groups to (c) DMI and (d) 4-OI groups relative to expression without either treatment (n = 3). Western blotting data showing differences in myogenic transcription factor expression between DM treated groups and groups treated with DM plus (e) DMI and (f) 4-OI over a 4 day time course as well as groups which were not exposed to either treatment for any period. (g) MYHC staining at day 4 of differentiation. Bar: 100 μm. Relative myogenic index (percentage of total nuclei associated with myotubes) of (h) DMI and (i) 4-OI. *** denotes p < 0.001, ****p < 0.0001 based on two-way ANOVA/t-test.
To investigate a role of physiological itaconate, C2C12 myoblast cells were then treated 98 with DM only or DM plus itaconate and then examined over 4 days. It was reported that 99 exogenous itaconate readily enters cells (9). Itaconate was used to portray physiological 100 mechanisms more accurately. Transcription levels of Myh2 and Myh7 were not 101 significantly impacted by itaconate exposure. However, the blunted induction of Myh1, 102 Tnnt1, and Tnnt3 was significant on the 4th day of exposure (Fig. 2a). Significant inhibition 103 of Myog transcription on days 2 and 4 was also observed in itaconate groups (Fig. 2b).

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Overall DMI shows the strongest myogenic inhibitory response and itaconate has 107 the weakest amount of myogenic suppression among itaconate and derivatives tested.

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Taken together, these results suggest that itaconate and its derivatives are sufficient to 109 inhibit myogenesis in C2C12 cells, modulating myogenic regulating factors at the 110 transcription and protein levels. inhibition was more severe than itaconate but not as extreme as the inhibition caused by 120 itaconate derivatives. In parallel, a potent and specific inhibitor of SDH, which is 121 Harzianopyridone (Harz) possessing antibiotic and antifungal effects (13), was tested to 122 determine whether this specific shared inhibition was the primary inducer of myogenesis 123 repression. We examined C2C12 cells exposed to Harz using the same methods to

Fig. 2. Itaconate represses C2C12 myogenesis by modulating myogenic transcription mechanisms.
(a) 4-day time course showcasing fold change differences of myogenic gene transcription levels detected via RT-qPCR when exposed to differential media with or without itaconate (7.5 mM) relative to the untreated group (n = 3). (b) Fold change of Myog mRNA expression at various time points comparing DM control groups to DM and itaconate groups relative to expression without either treatment (n = 3). (c) Western blotting data showing differences in myogenic transcription factor expression between DM treated groups and groups treated with both DM and itaconate over a 4 day time course as well as groups which were not exposed to either treatment for any period. (d) MYHC staining at day 4 of differentiation. Bar: 100 μm. (e) Relative myogenic index (percentage of total nuclei associated with myotubes). * denotes p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 based on two-way ANOVA/t-test.   Fig. 3. Malonate and pharmacological inhibitor of succinate dehydrogenase repress C2C12 myogenesis via similar mechanism of action to that of itaconate and its derivatives. Heatmap depiction of average fold change (Sig. denotes statistical differences at day 4, n = 3) in myogenic gene transcription levels detected via RT-qPCR when exposed to differential media with or without (a) dimethyl malonate (DMM: 5 mM) and (b) harzianopyridone (Harz: 4 µM) relative to the untreated group over a 4-day time course. Fold change of Myog mRNA expression at various time points comparing differentiation media (DM) control groups to (c) DMI and (d) 4-OI groups relative to expression without either treatment (n = 3). Western blotting data showing differences in myogenic transcription factor expression between DM treated groups and groups treated with DM plus (e) DMM and (f) Harz over a 4 day time course as well as groups which were not exposed to either treatment for any period. (g) MYHC staining at day 4 of differentiation. Bar: 100 μm. Relative myogenic index (percentage of total nuclei associated with myotubes) of (h) DMM and (i) Harz. * denotes p < 0.05, **p < 0.01, ****p < 0.0001 based on two-way ANOVA/t-test.

Fig. 4. Elevated exposure to succinate elicits inhibition of C2C12 myogenesis via mechanism similar to those observed when exposing cells to itaconate and malonate. (a)
4-day time course showcasing fold change differences of myogenic gene transcription levels detected via RT-qPCR when exposed to differential media with or without diethyl succinate (DES: 5 mM) relative to the untreated group (n = 3). (b) Fold change of Myog expression at various time points comparing DM control groups to DM and DES groups relative to expression without either treatment (n = 3). (c) Western blotting data showing differences in myogenic transcription factor expression between DM treated groups and groups treated with both DM and DES over a 4 day time course as well as groups which were not exposed to either treatment for any period. (d) MYHC staining at day 4 of differentiation. Bar: 100 μm. (e) Relative myogenic index (percentage of total nuclei associated with myotubes). * denotes p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 based on two-way ANOVA/t-test.
immunomodulation properties. It is a decarboxylated cis-aconitate which is primarily

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While seemingly propitious as a potential treatment, the fact is that knowledge is very 177 limited on the effects of itaconate and its derivates outside of those relating to the 178 immunomodulatory axis. A consequence of itaconate derivative use may be one or more 179 harmful side effects which could lessen enthusiasm for their utilization as treatments.
Blocking itaconate signaling, which has yet to be proposed, might also be used to 181 attenuate symptoms of other conditions.