The Role of miRNA Families on the Regulation of Skeletal Muscle Mass and Function Throughout the Murine Lifespan
Benard, Townsend.
2020
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Thesis
(Ph.D.)--Tufts University, 2020.
Submitted to the Dept. of Biochemical and Molecular Nutrition.
Advisor: Roger Fielding.
Committee: Donato Rivas, Sarah Lessard, Stefania Lamon-Fava, and Laurence Parnell.
Keyword: Nutrition.
Age-associated changes in the expression of skeletal muscle microRNA (miRNA), small (~18-22 nt) non-coding RNA ... read morefunctioning primarily in post-transcriptional regulation of gene expression, have been proposed as a potential contributor to sarcopenia, the age-associated loss of muscular strength, mass and/or function. Despite this, the progression of these changes across the lifespan are unclear. Similarly, the consistency of age-associated changes within miRNA families, groups of miRNAs targeting similar genes due to conservation of seed sequence, are largely uncharacterized. Understanding how broad changes in miRNA expression late within the lifespan contribute to the development and manifestation of anabolic resistance could provide insight into potential mechanisms for the maintenance of muscle mass and strength within aging. As a result, two investigations were conducted to translate age-associated changes in skeletal muscle miRNA family expression to targeted in-vitro-based investigations exploring the implication of such changes on critical anabolic signaling components and the phenomenon known as anabolic resistance within skeletal muscle. In the first investigation we quantified the expression of 752 distinct miRNA species in gastrocnemius muscles extracted from young (6 mo), middle-aged (18 mo), old-aged (24 mo) and advanced-aged (30 mo) C57BL/6 mice (n = 6-7/group) to assess the conservation and temporality of age-associated changes both within and between miRNA families. Individual miRNA and miRNA families demonstrating age-associated increases in expression exhibited large heterogeneity in the temporality of such changes. In contrast, age-associated reductions in highly abundant miRNA species occurred predominately within old and advanced age, a pattern highly conserved within miRNA families. Despite this conservation, age-associated reductions in miR-30-5p expression did not induce age-associated increases in Tnrc6a, an experimentally validated target gene. These data suggest conservation of regulatory mechanisms both within and between miRNA families alongside a limited role for individual miRNA families in the regulation of age-associated changes in target gene expression. In the second investigation we manipulated the expression of miR-30c-5p and Tnrc6a within C2C12 myoblasts to explore their respective role mediating mTORC1 anabolic signaling and the response to nutritive (L-leucine) and non-nutritive (IGF-1) anabolic stimuli. Both L-leucine and IGF-1 induced reductions in miR-30-5p abundance following acute treatment. Knockdown of Tnrc6a did not impair L-leucine or IGF-1-induced changes in p-Rps6 or p-4E-BP1, suggesting a limited role for Tnrc6a in anabolic signaling. In contrast, overexpression of miR-30c-5p potentiated p-Rps6 both basally and in response to L-leucine, in accordance with prior positive association between miR-30-5p, protein synthesis and skeletal muscle hypertrophy. Interestingly, inhibition of miR-30c-5p did not impair p-Rps6 or p-4E-BP1 either basally or in response to L-leucine and actually potentiated IGF-1-induced increases in p-Rps6. These findings demonstrate a stimuli-specific, Tnrc6a-independent relationship between miR-30c-5p and mTORC1 signaling. Taken together, these investigations demonstrate the need for a shift from characterizing individual miRNA:target gene relationships to broader miRNA:mRNA coregulatory networks. Future investigations should also look to explore conserved drivers of late-logistic changes within the skeletal muscle transcriptome.read less - ID:
- kh04f399p
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