NOS-dependent “On/Off” switch and apoptosis in the freshwater and aestivating lungfish P. annectens: skeletal vs cardiac muscle

African lungfishes (Protopterus spp.) are obligate air breathers, which aestivate during the dry season, undergoing prolonged skeletal muscle immobilization and cardiac re-adjustments. The molecular mechanisms underpinning aestivation and post-arousal recovery responses of cardiac and skeletal muscle are unknown. Using biochemical and immunofluorescence methods, we analysed expression, localization and activity of the endothelial NOS isoform, eNOS, and its partners, Akt and Hsp-90, in cardiac and skeletal muscles of Protopterus annectens under three experimental conditions: freshwater (FW), six months of aestivation (6mAe) and six days after arousal (6mAe6d). The modulation of a functional eNOS/NO system in these two muscle tissues in response to environmental stress was tissue specific. During aestivation, p-eNOS/eNOS and p-Akt/Akt ratios increased in the cardiac muscle and decreased in the inactive skeletal muscle, although Hsp-90 increased in both tissues. Analysis of cell turnover (TUNEL) showed that the apoptotic nuclei in the cardiac muscle remained comparable between the three conditions, but there was an increase in TUNEL positive nuclei in the skeletal muscle during aestivation. Expression of apoptosis repressor with caspase recruitment domain (ARC) was strongly reduced in the skeletal muscle of 6mAe fish, but remained unchanged in the cardiac muscle of 6mAe and 6mAe6d fish in comparison with the FW, indicating a preserved cardiac activity during aestivation. In conclusion, our results indicate the importance of the eNOS/NO system and cell turnover regulation in the morpho-functional re-adjustments of the cardiac and skeletal muscles of P. annectens during the switch from freshwater condition to aestivation and from aestivation to arousal.