Re: Fast And Furious Murgia Version
Frank Belair
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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.
The basic helix-loop-helix (bHLH) family of myogenic regulatory factors (MRFs) comprises four members, MyoD, myogenin, myogenic factor 5 (Myf5) and MRF4, which play key roles in skeletal muscle commitment and differentiation1. The MyoD and Myf5 genes are involved in muscle commitment during embryogenesis, whereas myogenin has a crucial downstream role in the differentiation of committed muscle progenitors into myofibres. Mrf4 differs from the other family members in that it has a biphasic pattern of expression during mouse development2. Mrf4 is transiently expressed at the same time as Myf5 at the onset of myogenesis in the embryo3 and can function as a determination gene, as some myogenesis takes place in a double Myf5/MyoD mutant in which Mrf4 is not compromised4. A later phase of Mrf4 expression starts during fetal development and continues throughout postnatal stages and is by far the predominant MRF expressed in adult muscle fibres5. However, the function of MRF4 in adult muscle is not known.
Short hairpin RNA (shRNA) sequences targeting Mrf4 mRNA were inserted into pSUPER plasmids and co-transfected in to cultured HEK-293 cells together with a plasmid encoding myc-tagged rat MRF4. A vector containing shRNA sequences targeting LacZ was used as a negative control. Two Mrf4-specific shRNAs, referred to as M1 and M2, were found to markedly decrease the expression of MRF4 (Supplementary Fig. 1a) and were thus selected for in vivo studies. Plasmids coding for M1 and M2 were then electroporated in to rat muscles, together with a plasmid encoding GFP. A marked decrease in nuclear staining for the endogenous MRF4 was seen in transfected muscle fibres, identified by GFP expression, compared with untransfected fibres within the same muscles (Supplementary Fig. 1b). Unlike MyoD and myogenin, which are prevalent in fast or slow muscles, respectively, we found that MRF4 is expressed at similar RNA and protein levels in the fast extensor digitorum longus (EDL) and slow soleus (SOL) muscles (not shown), in agreement with previous studies6,7. Therefore, we examined the effect of M1 and M2 in both EDL and SOL muscles.
The most obvious change induced by MRF4 knockdown was the marked hypertrophy of most transfected fibres compared with LacZ shRNA controls (Fig. 1a,b) and to non-transfected fibres in the same muscle (Fig. 1a and Supplementary Fig. 2). Muscle fibre hypertrophy was evident at 7 and 14 days post transfection in both innervated and denervated muscles, denervation atrophy being prevented by Mrf4 RNAi (Fig. 1b and Supplementary Fig. 3). In contrast, muscle fibre size was unaffected by overexpression of Mrf4 in adult muscles (Fig. 1c). We also examined the effects of Mrf4 knockdown and overexpression in regenerating muscles. Regenerating muscle growth was strikingly accelerated by Mrf4 knockdown, with fibre size more than doubled compared with controls (Fig. 1d and Supplementary Fig. 4). A smaller but significant change in the opposite direction was induced by Mrf4 overexpression in regenerating muscle, with fibre size being reduced by about 20% compared with control (Fig. 1e).
To validate the specificity of our RNAi experiments and rule out the possibility that the observed changes were due to off-target effects, we performed rescue experiments with RNAi-resistant target genes. The sequence recognized by M1 shRNA in rat Mrf4 has a single base difference in human Mrf4, so that expression of the human gene is not silenced by M1 in cultured HEK-293 cells (Fig. 1f). In vivo transfection experiments showed that the increase in fibre size induced by Mrf4 RNAi was completely abrogated when a plasmid encoding human Mrf4 was co-transfected with M1 (Fig. 1f). A similar rescue experiment with identical results was performed with M2, using mouse Mrf4, which is M2-resistant because of a two-base difference in the sequence recognized by M2 shRNA (Fig. 1g). Next, we asked whether the effect of Mrf4 knockdown on muscle growth is specific for this member of the MRF family and tested the effect of shRNAs targeting myogenin. However, no effect on muscle fibre size was observed when myogenin-specific shRNAs were transfected in to adult skeletal muscle (Fig. 1h), in agreement with the finding that muscle weight was unchanged by deletion of the myogenin gene in adult innervated muscles using an inducible knockout model8.
Muscle hypertrophy is always accompanied by increased protein synthesis9. To monitor protein synthesis in transfected muscles, we used a procedure based on the incorporation of puromycin into nascent peptides10. As shown in Fig. 1i, Mrf4 RNAi induced a significant increase in the amount of puromycin-labelled peptides compared with LacZ RNAi controls. This finding shows that protein synthesis is markedly increased during muscle hypertrophy induced by loss of MRF4, as in other models of muscle hypertrophy.
To address the mechanism of muscle hypertrophy induced by Mrf4 knockdown, we performed microarray analysis of innervated and denervated SOL muscles transfected with shRNA to Mrf4 (M1 sequence) and compared them with muscles transfected with shRNA to LacZ and examined after 7 days. We first examined differentially expressed genes in the four experimental groups and found that Mrf4 RNAi increased the expression of 677 genes and decreased the expression of 782 genes compared with the control LacZ RNAi (fold change >2 and adj. P
Next, we studied the effect of MRF4 on a MEF2-dependent reporter. In HEK-293 cells, the reporter was transactivated by myogenin, in agreement with previous studies11, but was inhibited by MRF4 (Fig. 4b). In adult skeletal muscle, the MEF2 reporter was likewise transactivated by myogenin but strongly inhibited by MRF4 overexpression (Fig. 4c). An opposite effect was induced by MRF4 knockdown: MEF2 reporter activity was markedly increased by Mrf4 shRNA (M1), whereas it was unaffected by shRNA against myogenin (Fig. 4d). A similar increase in MEF2 reporter activity was induced by M2 shRNA to rat MRF4 and this effect was completely abrogated by co-transfection with the RNAi-resistant mouse Mrf4 (Fig. 4e). The increase in MEF2 transcriptional activity induced by MRF4 knockdown was seen in both SOL and EDL muscles and was also obvious in denervated muscles, which showed decreased MEF2 activity relative to control muscles (Supplementary Fig. 7).
To determine whether a specific MRF4 domain is involved in the repression of the MEF2 reporter, we transfected both SOL and EDL muscles with chimeric transgenes containing either the MRF4 N-terminal domain linked to myogenin bHLH and C-terminal domains, or the myogenin N-terminal domain linked to MRF4 bHLH and C-terminal domains (Fig. 4f). Whereas the response to the first chimera is similar to that of myogenin, the second chimera, containing the MRF4 C-terminal domain, has a repressive effect on the MEF2 reporter, similar to that of MRF4 (Fig. 4g). These results suggest that the MRF4-dependent repression of MEF2 transcriptional activity in adult skeletal muscle is associated with the C-terminal domain of MRF4.
To determine whether MEF2 is required for mediating myofibre hypertrophy induced by Mrf4 RNAi, we used a truncated dominant negative MEF2 (dnMEF2, Fig. 5a), which abrogates MEF2-dependent transcriptional activity in cardiac muscle of transgenic mice12 and inhibits muscle-specific gene expression and myotube formation in cultured skeletal muscle cells13. The increase in fibre size induced by Mrf4 RNAi was prevented by co-transfection with dnMEF2 in both innervated and denervated muscles (Fig. 5b). This experiment supports a necessary role of MEF2 in mediating the effect of Mrf4 RNAi and at the same time supports the existence of a direct link between MEF2 activity and muscle fibre growth in adult skeletal muscle. The following experiment was aimed at validating the existence of this link.
An inducible, constitutively active MEF2 (caMEF2) was used, together with a DNA-binding-deficient version of this construct (Δ-caMEF2), to determine whether an increase in MEF2 transcriptional activity is sufficient to induce myofibre hypertrophy in adult skeletal muscle (Fig. 5a). In agreement with studies in cultured neurons14, we found that caMEF2 was localized in myonuclei of transfected myofibres in animals treated with tamoxifen (Fig. 5c). Both innervated and denervated myofibres transfected with caMEF2, but not those transfected with Δ-caMEF2, showed a significant hypertrophy after treatment with tamoxifen (Fig. 5d). In animals treated with oil vehicle rather than tamoxifen, caMEF2 remained sequestered in the cytoplasm and fibre size was unchanged (Fig. 5e,f). These results indicate that MEF2 transcriptional activation is sufficient to induce myofibre hypertrophy in adult skeletal muscle.
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