The Exon Junction Complex proteins are non‐AMP kinase dependent sensors of energetic stress and regulate contractility and sarcomere structure in cardiac myocytes

Abstract

The heart is known to undergo significant alterations in gene expression during stress and sometimes this adaptation can lead to heart failure. The mechanism by which cells select specific mRNAs for translation is not fully understood, but the Exon Junction Complex (EJC) plays a major role in this process in eukaryotes. The EJC selects the mRNA isoforms to be exported to the cytoplasm and determines whether they are then translated into protein. In this study we hypothesised that the EJC is involved in the regulation of gene expression during the stress response in cardiac myocytes. Using cultured rat neonatal myocytes and subcellular fractionation we examined the cellular distribution of two major components of the EJC in response to metabolic stress. In cardiac myocytes we found that there was a significant relocalisation of eIF4A3 and mago from the nucleus to cytoplasm following 18 hours of hypoxia. To examine the effect of metabolic stress on the EJC in more detail we treated cardiac myocytes with 50mM NaN3 for 4 hours to mimic the metabolic stress induced by hypoxia. Following NaN3 treatment there was a significant relocalisation of eIF4A3 and mago to the cytoplasm and this was associated with the formation of cytoplasmic stress granules. However, immunocytochemical staining with the stress granule marker PABP1 showed that cytoplasmic mago did not colocalise with stress granules. To examine whether the effects of metabolic stress on the EJC proteins was dependent on the metabolic sensor AMP kinase (AMPK), we treated myocytes with 1μM dorsomorphin in combination with NaN3. Dorsomorphin augmented the translocation of mago and eIF4A3 from the nucleus to the cytoplasm. This indicates that the EJC proteins respond to metabolic stress independently of AMPK activity. Moreover, the inhibition of AMPK was associated with increased metabolic cellular stress resulting in the augmented subcellular translocation of EJC proteins. Constructs of eIF4A3 tagged with green fluorescence protein on the N terminus resulted in a predominantly cytosolic protein distribution that did not relocalise in response to NaN3 treatment. This suggests that the N‐terminus of eIF4A3 is necessary for subcellular localisation. Finally, to determine the function of eIF4A3 in cardiac myocytes we knocked down the protein with siRNA. Protein knockdown of eIF4A3 resulted in cessation of cell contractility 96 hours post treatment. There was also a significant reduction in the number of intact sarcomeres suggesting that the contractile filaments were degrading in response to reduced eIF4A3. Taken together, these data suggest that eIF4A3 and other EJC members play an important role in the myocyte stress response, cell contractility and morphology.Support or Funding InformationThis work was supported by the British Heart Foundation project grant PG/10/64/28520 and PG/10/97/28666