Exercise Related microRNAs in C. elegans Regulate Calcium Homeostasis and Mitochondrial Dynamics: Conserved pathways, Divergent microRNAs

In this study, a 5-day exercise programme that has been demonstrated to increase mitochondrial content and improve healthspan was performed in C. elegans. Small RNA sequencing of miRs, revealed changes in specific miRs following exercise. Specifically, there were increased levels of cel-miR-57-5p and cel-miR-249-3p, whereas cel-miR-72-3p and cel-miR-77-5p decreased in response to exercise. Furthermore mir-57 and mir-249 mutant strains had enhanced fertility, survival and lifespan, whereas mir-72 and mir-77 mutant strains had diminished fertility, survival and lifespan. Interestingly both mir-77 and mir-249 mutant strains exhibited impaired mitochondrial morphology and reduced fitness following exercise. cel-miR-77-5p targets a number of genes regulating Ca2+ homeostasis such as ipp-5 (inositol Polyphosphate-5-phosphatase), sca-1 (Ca2+ transporting ATPase) and ncx-2 (mitochondrial Na+/Ca2+ antiporter). Although the exercise related miRs identified in C. elegans were not conserved in mammals. Exercise related mammalian miRs identified from the literature: mmu-miR-181a-5p, mmu-miR-199a-5p and mmu-miR-378a-3p, shared targets with the exercise related miRs identified in C. elegans. The levels of mmu-miR-181a-5p, mmu-miR-199a-5p and mmu-miR-378a-3p increased in response to an acute physiological bolus of H2O2 in an in vitro myoblast model. Treatment of myoblasts with synthetic mmu-miR-181a-5p (miR181) and mmu-miR-378a-3p (miR378) led to enhanced mitochondrial content, autophagy markers and improved myogenesis. Among the shared target genes and pathways were the regulation of Ca2+ homeostasis and mitochondrial associated genes. Specifically, the results demonstrate that mmu-miR-181a-5p and cel-miR-77-5p commonly target Inositol Polyphosphate-5-Phosphatase A (Inpp5a)/ipp-5 that reduces inositol 3 phosphate levels (IP3), contributing to the regulation of Ca2+ metabolism in muscle. Ca2+ imaging was performed in myotubes following treatment with miR181 and AntagomiR-181a (AM181), revealing decreased Ca2+ flux with AM181 and elevated Inpp5a. Furthermore, mmu-miR-378a-3p and cel-miR-249-3p share a putative target, Kinase Suppressor of Ras (Ksr1)/ksr-2, involved in activating the Mitogen activated protein kinase (MAPK) pathway and mitochondrial biogenesis. The data indicates inter-species conservation in potential targets of exercise related miRs, providing insights into likely conserved regulatory mechanisms in cellular processes related to Ca2+ homeostasis and mitochondrial function.