Myonuclear identification and epigenetics in skeletal muscle

Abstract

Skeletal muscle is a heterogeneous tissue comprised of a variety of different cell types, including the multinucleated skeletal myofiber. Although myofibers are post-mitotic, they hold a remarkable ability to adapt new functional characteristics. One of the mechanisms used to achieve phenotypical plasticity is by structural reorganization of the genome. Commonly referred to as epigenetics, these adaptations occur through different processes such as methylation and acetylation of histones. In this thesis, we wanted to study the epigenetic landscape to understand more about the mechanisms of muscle physiology. In paper I, we investigate if myonuclei are lost from the myofiber during cancer-induced atrophy. We induced cachexia by transplanting PC3 prostate cancer cells in nu/nu mice. Using in-vivo imaging techniques and histochemical analysis, we found no loss of myofibers or myonuclei from tissue that had undergone severe atrophy. In paper II, we address the issue of cellular heterogeneity and show that an antibody against Pericentriolar material-1 (PCM1) can be used to identify myonuclei on tissue cross-sections from mice, rats, and humans. The labeling allows us to distinguish myonuclei from other nuclei residing in the tissues. In paper III, we used the PCM1 antibody to enrich myonuclei for epigenetic analysis. By comparing data from sorted and unsorted nuclei, we show that myonuclear enrichment reduces interference from stromal cells. The method was furthermore used to generate global maps of histone modifications in the fast/glycolytic extensor digitorum longus (EDL) and slow/oxidative soleus (SOL) muscles and found that the differences in phenotype are reflected in the epigenetic landscape. The work presented in this thesis make it possible to study skeletal muscle fibers more specifically; in regards to physiological and epigenetic responses to a changing milieu without complicating responses from other cell types. By showing that myonuclei are preserved during cachexia, our model can be used to study the underlying causes of atrophy.