MRF4 negatively regulates adult skeletal muscle growth by repressing MEF2 activity

The myogenic regulatory factor MRF4 is highly expressed in adult skeletal muscle but its function is unknown. Here we show that Mrf4 knockdown in adult muscle induces hypertrophy and prevents denervation-induced atrophy. This effect is accompanied by increased protein synthesis and widespread activation of muscle-specific genes, many of which are targets of MEF2 transcription factors. MEF2-dependent genes represent the top-ranking gene set enriched after Mrf4 RNAi and a MEF2 reporter is inhibited by co-transfected MRF4 and activated by Mrf4 RNAi. The Mrf4 RNAi-dependent increase in fibre size is prevented by dominant negative MEF2, while constitutively active MEF2 is able to induce myofibre hypertrophy. The nuclear localization of the MEF2 corepressor HDAC4 is impaired by Mrf4 knockdown, suggesting that MRF4 acts by stabilizing a repressor complex that controls MEF2 activity. These findings open new perspectives in the search for therapeutic targets to prevent muscle wasting, in particular sarcopenia and cachexia.


Supplementary Figure 2. Fiber size changes in transfected versus non-transfected fibers in the same muscle.
Transverse sections of soleus transfected with either LacZ shRNAs or Mrf4 shRNAs (M1) and examined at 14 days after transfection. Transfected fibers identified by GFP fluorescence, section stained for dystrophin (red) to visualize fiber profiles. Note larger fiber size in muscle fibers transfected with Mrf4 shRNAs compared to neighboring untransfected fibers. Scale bar 100 µm.

Supplementary Figure 3. Effect of Mrf4
RNAi in adult rat muscle at 14d after transfection. Crosssectional areas of muscle fibers in innervated and denervated SOL and EDL muscles transfected with M1 or M2 and co-transfected with GFP. Values normalized to fibers transfected with LacZ shRNAs in innervated SOL or EDL. At least 4 muscles per group were analyzed, data are presented as means±SEM from at least three independent experiments. Statistical analysis was performed using Student's two-tailed t-test (*p<0.05, **p<0.01, ***p<0.001).

Supplementary Figure 4. Effect of Mrf4
RNAi in regenerating muscle. Rat SOL was cotransfected at day 3 after bupivacaine-induced muscle injury with GFP and Mrf4 or LacZ shRNAs and examined 7 days later. Serial sections were stained with H&E, and with anti-GFP (to visualize transfected fibers), anti-dystrophin (to visualize fiber profiles), Hoechst (to visualize nuclei), and anti-MyHC-emb. Scale bar 100 μm.

Supplementary Figure 5. Effect of Mrf4
RNAi on global gene expression profile. Heatmap representing hierarchical clustering of differentially expressed genes (p-value < 0.05; fold change > 2) between Mrf4 RNAi (M1) and LacZ RNAi innervated and denervated muscles. Each lane corresponds to one animal, 3 animals per group were analyzed.

Supplementary Figure 6. Changes in protein levels of selected sarcomeric proteins induced by
Mrf4 RNAi. SOL muscle transfected with Mrf4 shRNAs and examined at 7 days after transfection by immunofluorescence with anti-MyHC slow, antiMyHC 2B anti-cardiac troponin antibodies. Note that MyHC-2B and troponin I, whose transcripts are upregulated by Mrf4 knockdown (see Fig. 2c and Supplementary Table 1), are not detectable at the protein level in transfected muscle fibers. Scale bar 100 µm.

Supplementary Figure 7. The increase in MEF2 transcriptional activity induced by Mrf4 knockdown (M1) is seen in both innervated and denervated SOL and EDL muscles.
Muscles were transfected with a MEF2 reporter and analyzed after 7 days. Data are expressed as percent luciferase activity relative to control innervated muscles. Note that MEF2 reporter activity is reduced in denervated compared to innervated muscles especially in the EDL muscle. Data are presented as means±SEM from at least three independent experiments (n=4). Statistical analysis was performed using Student's two-tailed t-test (*p<0.05, **p<0.01, ***p<0.001).

Supplementary Figure 8. Effect of a GAL4-MRF4 chimera on GAL4 DNA-binding domain-luciferase
reporter. HEK 293 cells were co-transfected with a luciferase reporter, driven by five GAL4 binding sites upstream the TK minimal promoter, and either the basic GAL4 DNA-binding domain as a control or constructs encoding fusion proteins containing MRF4, VP16 or SAP30 cDNAs cloned in frame to the GAL4 DNA-binding domain. Data are expressed as percent luciferase activity relative to control and are presented as means±SEM from at least three independent experiments (n=4). Statistical analysis was performed using Student's two-tailed t-test (*p<0.05, **p<0.01, ***p<0.001). promoters. MRF4 binding was determined by ChIP-qPCR in denervated gastrocnemius muscle of adult wild type (WT) and Mrf4 knockout (Mrf4 KO) mice and expressed relative to IgG binding. An intronic region of Hprt was used as negative control. Data are presented as means±SEM from at least three independent experiments (n=3). Statistical analysis was performed using Student's two-tailed t-test (*p<0.05, **p<0.01, ***p<0.001). Figure 11. Uncropped scans of Western Blots shown in Fig. 1, Fig. 4, Fig. 6 and Supplementary Fig. 1. Changes in muscles transfected with shRNAs against MRF4 compared to muscles transfected with shRNAs against LacZ. Pooled data from innervated and denervated muscles. Threshold: