Transcriptome Analysis Reveals Regulation of Novel lncRNAs Transcribed Near Contractile Protein‐Coding Genes During Skeletal Muscle Unloading
Authors: Resnick Jessica D. , Lowrey Angela J. , Akers Robert G. , Theodore Lindsey A. , Wiles Amy M. , Pandorf Clay E. 
Published: November 2023

Unloading of skeletal muscle induces atrophy and a shift in slow fiber‐type muscles toward a fast‐fiber phenotype. This remodeling results in altered expression of contractile protein‐coding genes. It has become recognized that long noncoding RNAs (lncRNAs) are widely expressed throughout the genome and many have been shown to play a significant role in the transcriptional regulation of protein‐coding genes. The purpose of this study was to examine the transcriptome response to seven days of hindlimb unloading suspension (HS) in adult rat soleus (SOL) muscle using next generation sequencing (RNA‐Seq). A normal ambulatory group of rats served as the controls (CON); (N=6). We hypothesized that unannotated lncRNAs are differentially expressed during the phenotype shift and that they may regulate protein‐coding genes. Raw RNA‐Seq data was processed and analyzed using the Tuxedo suite. Analysis of the transcriptome data led to the selection of a subset of slow‐phenotype contractile protein‐coding genes that were also associated with the differential expression of novel lncRNAs transcribed within 10kb of the coding genes. Transcription of the lncRNAs was validated with RT‐PCR and their abundance was assessed in both the sense (S) and antisense (AS) orientations, relative to the neighboring S coding gene, by using gene‐specific primers during the RT.

The following selected slow‐phenotype contractile protein‐coding genes were downregulated with HS as compared to CON SOL and were associated with upregulation of an lncRNA in the AS orientation: Troponin I Type 1 (TNNI1), Troponin C Type 1 (TNNC1), Myosin Heavy Chain 7/slow type 1 (MYH7), and Myosin Regulatory Light Chain 2 (MYL2). RT‐PCR was performed to target multiple locations in the lncRNA in CON and HS SOL. We report the following representative results of one specific region for each lncRNA. A decrease in TNNI1 mRNA in response to HS was associated with a 2.7‐fold (p<0.05) increase in an AS lncRNA assessed ~2.2kb from the 5′‐end of TNNI1. Downregulation of TNNC1 mRNA with HS was associated with a 1.7‐fold (p<0.05) increase in an AS lncRNA as measured ~2.7kb from the 3′‐end of TNNC1. A reduction in MYH7 mRNA with HS occurred with a 2.1‐fold increase (p<0.05) in AS lncRNA, as measured 10kb from the 5′‐end of MYH7. A decline in MYL2 mRNA with HS was associated with a 12‐fold increase (p<0.05) in AS lncRNA, as measured 9kb from the 5′‐end of MYL2. Further work will be required to delineate the function of these lncRNAs. In summary, we report on the discovery of four lncRNAs that are upregulated in response to muscle disuse unloading in conjunction with downregulation of adjacent contractile protein‐coding genes. The expression patterns are consistent with an inhibitory role of the AS lncRNAs on the protein coding genes. The divergent transcription of these newly identified lncRNAs may indicate their involvement in the muscle unloading‐induced coordinated shift in expression from slow to fast contractile proteins.

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Medcen Community Health Foundation & Mercer University School of Medicine