Long non-coding RNAs expressed in fast gycolytic and slow oxidative myofibers

Long non-coding RNAs (lncRNAs) are emerging as important players in the regulation of several aspects of cellular biology. For a better comprehension of their function it is fundamental to determine their tissue or cell specificity and to identify their subcellular localization. In fact, the activity of lncRNAs may vary according to cell-type specific expression and subcellular localization. Myofibers are motor units of skeletal muscles characterized by great metabolic plasticity. How lncRNAs are expressed in different myofibers, participate to metabolism regulation, and are compartmentalized within a single myofiber is still unknown. We compiled a complete and integrated catalogue of lncRNAs expressed in skeletal muscle, associating the fiber-type specificity and subcellular location to each of them, demonstrating that many are altered when muscles changes myofiber composition and metabolism according to specific stimuli. We demonstrated that the lncRNA Pvt1, activated early during muscle atrophy, impacts mitochondrial respiration and morphology and affects mito/autophagy and myofiber size in vivo by binding specific DNA regions. This work corroborates the importance of lncRNAs in the regulation of metabolism and neuromuscular pathologies and offers a valuable resource to study the metabolism in single cells characterized by pronounced plasticity. EDL and soleus mouse muscles were incubated with type I collagenase (Sigma-Aldrich) to dissociate intact myofibers that were separately collected under stereomicroscope. Isolated myofibers were divided in two parts: one was immersed in Laemmli buffer for fiber typing; the other was placed in TRIzol Reagent (Life Technologies) for RNA purification. Myofibers were classified according to their myosin heavy chain (MyHC) protein isoform in pure MyHC-1, and -2B. We analyzed the transcription profiles of 10 single isolated myofibers (biological replicas) for type 2B myofibers and 11 single isolated myofibers (biological replicates) for type 1 myofibers (total: 21 experiments). One-color hybridizations were performed on SurePrint G3 Mouse GE 8x60K Microarrays (Agilent Technologies). Microarray platform was re-annotated according transcripts in Ensembl 74. Only long non coding RNAs were considered for expression analyses.