Skeletal muscle has a remarkable ability to rapidly remodel to meet physiological demands. As such, it is one of the most adaptable tissues in the human body, and this adaptability is extraordinarily well conserved through evolution. Among the many types of muscle remodeling, the rapid growth of muscle to increased load (hypertrophy) remains one of the most dramatic and best studied.
We sought to define the very acute responses of myofibers to a hypertrophic stimulus, focusing on both the proteomic changes to abundant myofibrillar proteins, as well as alterations in splicing of these same large structural proteins at the mRNA level. Muscle structural genes and proteins are amongst the largest and most complex in the mammalian genome, making them a challenge to study. Given the importance of structural proteins to muscle function, we applied emerging technologies to learn about how functional load affects expression of structural proteins and transcript splicing of structural genes.
The experimental model we used was synergist ablation hypertrophy surgery in the mouse. We show that this provided a controlled acute and robust hypertrophic response, with a probability of causing acute inflammation and long-term muscle injury. Our results suggest that acute inflammation in this model is caused by both the surgery and functional overload. However by stratifying the muscles by neutrophil infiltration, we were able to select muscles with mild inflammation. Using this animal model, we designed a quantitative proteomic time-series to capture the immediate changes (within hours) of the initial hypertrophic stimulus using stable isotope methods (SILAM). To study the transcriptional changes in these very large structural genes, we employed a relatively new PacBio long-read RNA isoform sequencing technology to catalog novel transcripts and splice differences between muscles of different fiber types.
By time-series proteomics analysis of our hypertrophy model we report changes in muscle structural proteins within hours post-stimulus. Our key finding was a –2.36 fold down-regulation of Titin over the course of 6–24 hours after functional overload. These results validate our quantitative proteomics approach as a useful method to capture immediate changes in structural proteins levels, but identify the need to further improve muscle hypertrophy models to reduce variation in the muscle response and thus increase the sensitivity of detecting protein that are targeted by hypertrophic stimuli. Therefore, we supplemented these proteomic results with transcriptional analysis of splice isoforms of muscle structural genes. To our knowledge, there are no long-read RNA-Seq studies performed on muscle thus far. For this reason, we developed the Pacific Biosciences IsoSeq (isoform sequencing) method-based approach. We applied it to study structural protein transcript isoforms in three metabolically distinct healthy muscles: extensor digitorum longus (fast-twitch, glycolytic fibers), soleus (slow-twitch, oxidative fibers), and cardiac (cardiocytes). This allowed us to identify novel muscle structural protein transcripts unique to specific muscle tissues. Our approach identified 2,631 exons differentially used across 443 genes between the three muscle types. Twelve of the differentially used exons were found in eight muscle structural/sarcomeric genes, which likely affect the structure and function of each muscle type. We selected three out of the 12 exons from the list of muscle structural proteins for RT-PCR validation including: Titin, Nebulin and Nebulin related anchoring protein (Nrap). We found differential exon usage between skeletal (soleus and EDL) and cardiac muscles and an unannotated exon (e.g. Neb). Thus, using the IsoSeq method is both useful and feasible for splice isoform discovery in complex muscle transcripts.
In conclusion, we have used an acute time series of proteomic changes, as well as comparative long-range splicing of mRNAs, to identify the importance of changes in levels and alternative splicing of structural proteins in skeletal muscle remodeling and function. Our proteomic and transcriptomic methods can help serve as useful tools for the muscle field. Importantly, understanding the molecular mechanisms behind remodeling is relevant for many chronic muscle diseases, where a better description of disease and activity-dependent changes in muscle are much needed for development of novel therapeutics.
|Advisor:||Hoffman, Eric P.|
|Commitee:||Brown, Kristy J., Chen, Yi-Wen, Jaiswal, Jyoti, Partridge, Terence|
|School:||The George Washington University|
|Department:||Biochemistry & Systems Biology|
|School Location:||United States -- District of Columbia|
|Source:||DAI-B 79/08(E), Dissertation Abstracts International|
|Subjects:||Genetics, Systematic biology, Bioinformatics|
|Keywords:||Alternative splicing, Hypertrophy, Long-read RNA-Seq, Quantitative proteomics, Skeletal muscle, Synergist ablation|
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