Muscle memory is defined as the capacity of muscle to respond differently to environmental stimuli in an adaptive or maladaptive manner if the stimuli have been previously encountered. Whether and how this memory is associated to epigenetic changes remains still largely undetermined. Among all stimuli, decrease of oxygen availability (hypoxia) represents a prominent environmental stressor. Chronic hypoxia is a common feature in many cardiac diseases, such as heart failure, ischemic heart disease, stroke. Hypoxia is also very common in elite sport athletes that use hypoxic training to improve performance. Investigating whether and how hypoxia induces epigenetic memory in muscle cells might help to find therapeutic strategies for optimizing athletic, counteracting muscle wasting conditions and age-related muscle loss in the general population, understanding and treating cardiovascular diseases.

To address this question, we studied in contractile muscle cells the effect of repeated hypoxic stimulus on cell functions and DNA methylation state, by focusing on the insulin-like growth factor 2 (IGF2) and H19 region. These genes are regulated by an imprinting control DNA region (ICR), whose methylation status influences selective IGF2 expression to the detriment of the long non-coding RNA encoded by the downstream gene H19.

We used a murine myoblast cell line (C2C12) with high proliferative rate, ability to easily differentiate in myotubes and with skeletal and cardiac muscle features. Cells were exposed to normoxia (21% O2) or a first hypoxic stimulus (1% O2) during 24h or 48h, then cultured for 3 weeks (corresponding to 42 population doublings) and exposed to a second stimulus. ICR region methylation and IGFII and H19 expression were analysed by qRT-PCR et cell functions by flow cytometry.

We observed upon an initial hypoxic stimulus, a cell number decrease (P<0.0001*** vs normoxia), accompanied by a decreased ICR methylation (P<0.0001*** vs normoxia) and an upregulation of H19 (P<0.0001*** vs normoxia). After a second stimulus of either 24h or 48h, all these parameters were again significantly reduced compared to normoxia (P<0.0001***), but remarkably, significantly higher than after the first stimuli.

Our results show that a hypoxic stimulus induces epigenetic modification in C2C12 cells and that cells keep memory of the first stimulus by differently respond to a second injury.