In today’s #apaperaday, Prof. Aartsma-Rus reads and comments on the paper titled: Cytoplasmic HDAC4 regulates the membrane repair mechanism in Duchenne muscular dystrophy.
Today’s pick is from J of Cachexia Sarcopenia and Muscle by Renzini et al on the function of HDAC4 in muscle repair in a dystrophic state IN MICE. Doi 10.1012/jcsm.12891.
Histone deacetylases (HDACs) play a role in histone acetylation and epigenetic modifications. HDAC4 however belongs to class IIa HDACs which are less efficient histone deacetylators and instead act in other ways to inhibit transcription.
For instance, HDAC4 deacetylases cytoplasmic proteins such as myosin heavy chain and can regulate muscle metabolism in this manner. It also plays a role in injury repair. However, the function of HDAC4 is not fully elucidated, especially not in a dystrophic environment
To study this, the authors generated inducible HDAC4 knockout mice, where they could specifically knockout HDAC4 in muscle. Lack of HDAC4 does not lead to pathology by itself. Here authors crossed the inducible knockout model with the mdx model lacking dystrophin (dko mice).
Authors show that HDAC4 expression is increased in mdx mice and in Duchenne patients. It is reduced in the dko mice (KO in the graph) but not fully depleted. Authors confirmed there was no overcompensation of other HDACs in the dko mice.
Authors assessed muscle damage by injecting Evan’s blue dye, seeing more damage in DKO than mdx (no wild types included and n = 3 only). They observed a reduction in centrally located nuclei in DKO compared to mdx. This can mean less regeneration or less need for regeneration.
Given that authors showed more damage it means less capacity for regeneration. Running to exhaustion was reduced in dko compared to mdx especially at 16 months. Authors isolated myoblasts and showed in vitro that differentiation was less effective for dko than for mdx myoblasts.
Furthermore, survival of dko myoblasts was impaired compared to mdx. Speculating a role for HDAC4 in membrane repair authors transfected plasmids expressing membrane repair proteins dysferlin and Trim72. Only Trim72 improved survival of myoblasts.
Electroporation of Trim72 plasmids to gastrocnemius muscles in mice reduced damage after downhill running compared to GFP control plasmid and improved running times (the latter is surprising as only gastrocnemius muscles were treated and more muscles are involved in running – n=4.
Authors finally showed that it is particularly the cytoplasmiccytoplasmatic roles of HDAC4 that play a role in muscle repair by transfecting or electroporating HDAC4s that either are restricted to the cytoplasm or the nucleus. They further show that HDAC4 can stabilize Trim32 mRNA.
Authors conclude that their data confirms a role for HDAC4 in muscle repair and that increased levels of HDAC4 would be expected to improve pathology (rather than the inhibition that is done by HDAC inhibitors currently in clinical trials.
First some comments about the work: no wild types were used – however, the comparison between mdx and dko can still be made without this. Authors do not specify gender however, and use very small groups (3-4 animals often) – we cannot rule out a gender effect as a cofounder.
Despite this I do believe HDAC4 plays a role in muscle repair in dystrophic mice. Authors suggest that givinostat (an inhibitor of many HDACs, not specifically HDAC4) may not be effective in patients (it is currently in trials for Duchenne). Their reasoning is:
- HDAC4 should be increased rather than decreased
- HDAC inhibition was effective in young mice but not in adults
- No functional effects were seen in the phase 2 trial with givinostat in Duchenne.
- HDAC4 was studied here in isolation and only for its effect on muscle injury and repair. HDACs do much more, they also play a role in fibrosis formation and inflammation for instance. These processes were not studied here for HDAC4 (due to muscle restricted knockdown).
- Adult vs young mice: you cannot extrapolate this to the human situation one to one. Young mice have active pathology while adults do not. Patients have active pathology throughout life. So it is possible that there will be an effect also in older patients.
- The goal of study was not to show functional effects so it was not powered to do this. Lack of functional effects in a phase 3 trial would be a good argument, but that trial is still ongoing, so we do not know the results yet. We do know that in Becker patients givinostat treatment appeared to improve muscle quality measured by MRI.
One cannot compare givinostat (inhibiting many HDACs in multiple tissues) with local knockdown of HDAC4 in muscle. Note that I am now pinpointing one aspect of the discussion – most of the discussion is on the specific role of HDAC4 in muscle.
Pictures by Annemieke, used with permission.