α7β1 integrin regulation of skeletal muscle growth in response to mechanical stimulation

The α7β1 integrin has been proposed to serve as a mechanosensor and essential regulator of myofiber remodeling given its localization at the membrane and primary role in adhering the outer extracellular matrix to the inner actin cytoskeleton. However, additional work is necessary to affirm a primary role for the α7 integrin subunit in the regulation of skeletal muscle mass. The purpose of this dissertation was to 1) use gene expression profiling to reveal potential mechanisms by which the α7BX2 integrin can promote an increase in muscle mass post- eccentric exercise, 2) determine the extent to which the α7BX2 integrin contributes to an increase in muscle mass after chronic overload, and 3) determine the extent to which α7 integrin overexpression or integrin ligand (laminin-111, LM-111) binding can improve the anabolic response to loading in aged mice. Aim 1 used microarray analysis using RNA extracted from skeletal muscle of wild type (WT) or transgenic mice (MCK:α7BX2 integrin; α7Tg), under sedentary conditions and 3 hrs after an acute bout of downhill running. The results suggest that the α7β1 integrin initiates transcription of genes that allow for protection from stress and modulation of protein synthesis, both which may contribute to positive adaptations in skeletal muscle with mechanical stimulation. In Aim 2, WT and α7Tg mice were subjected to myotenectomy (MTE) of the gastrocnemius muscles and subsequent chronic mechanical loading (CML) for 1 day (1D) or 14 days (14D). The results suggest that the α7β1 integrin augments muscle mass and adaptation, and several changes may account for this observation, including a reduction in damage and stress, yes-associated protein (YAP) content, and increased phospho- 4E-BP1 on Ser65. In Aim 3, α7Tg overexpression was not sufficient to rescue the anabolic response to chronic loading in aged mice, yet injection of LM-111 significantly restored integrin localization at the membrane and the anabolic response to a mechanical stimulus. Overall, the studies in this dissertation suggest that the α7β1 integrin is an important regulator of muscle remodeling that leads to an increase in mass in response to mechanical strain, and that manipulation of the extracellular matrix within the microenvironment may provide a novel approach towards maintenance of mechanosensing and the anabolic response across the lifespan. These observations in mice provide impetus for assessment of the relationship between extracellular matrix protein composition and subsequent α7β1 integrin-mediated remodeling within human skeletal muscle

Investigation of a role for the α7 integrin as a mechanotransducer of hypertrophic signaling in skeletal muscle

The α7β1 integrin is a transmembrane protein in skeletal muscle which serves as a focal adhesion at costameres of skeletal muscle, providing a critical link between the actin cytoskeleton inside the cell with laminin in the basement membrane of the extracellular matrix. Previous research has demonstrated that overexpression of the α7 integrin accelerates the muscle growth response to eccentric exercise compared to wild type controls. PURPOSE: The goal of this project was to elucidate a role for the α7 integrin as a mechanotransducer and intrinsic regulator of hypertrophic signaling in response to mechanical strain. METHODS: Primary myoblasts extracted from wild type (WT) mice were developed into myotubes in culture to determine the hypertrophic signaling response to mechanical strain. Primary myoblasts from α7 knockout (KO) mice were used as controls for these experiments. Further experimentation employed transient transfection of WT myoblasts with a α7 integrin plasmid transgene containing a muscle creatine kinase and myosin heavy chain promoter (MHCK7) to induce α7 integrin expression. Transfected myotubes were subjected to overnight incubations in wortmannin with the goal of inhibiting any potential autocrine-mediated growth factor signaling pathway through PI3K inhibition. RESULTS: Lack of α7 integrin was verified in KO cells. In WT, mechanical strain increased α7 integrin protein at 3 PS on both substrates compared to control (P<0.05; 1.8-fold collagen, 3-fold laminin). Phosphorylation of p70 S6K was increased 5-fold only on laminin at 3 PS (P<0.05). In contrast, no change in p70 S6K phosphorylation was observed in KO cells on either substrate. Interestingly, mTOR phosphorylation was not significantly altered in WT myotubes, and no change was observed in KO myotubes in response to strain. Transgenic overexpression of the α7 integrin in myotubes resulted in a trend toward increased activation of hypertrophic signaling compared to controls, which did not reach statistical significance. CONCLUSION: This study provides evidence that the α7 integrin is upregulated in response to mechanical strain and is inconclusive on whether the α7 integrin is an intrinsic regulator of strain-induced hypertrophic signaling

Diet-induced obesity regulates adipose-resident stromal cell quantity and extracellular matrix gene expression

Adipose tissue expansion during periods of excess nutrient intake requires significant turnover of the extracellular matrix (ECM) to allow for maximal lipid filling. Recent data suggest that stromal cells may be a primary contributor to ECM modifications in visceral adipose. The purpose of this study was to investigate the capacity for high fat diet (HFD)-induced obesity to alter adipose-derived stromal cell (ADSC) relative quantity and ECM gene expression, and determine the extent to which exercise training can mitigate such changes. Male C57BL/6J mice were placed on control or HFD for 8 weeks prior to and following initiation of a 16 week treadmill exercise program. ADSCs (Sca-1+CD45−) were isolated from epididymal adipose tissue and mRNA was evaluated using high throughput qPCR. Stromal cells were also obtained from skeletal muscle (MDSC). HFD decreased the quantity of ADSCs and markedly altered gene expression related to ECM remodeling (Col1α1, MMP2, MMP9, Timp1). Exercise did not reverse these changes. MDSCs were minimally altered by HFD or exercise. Overall, the data from this study suggest that ADSCs decrease in quantity and contribute to adipose ECM remodeling in response to obesity, and exercise training does not significantly impact these outcomes

Reversal of deficits in aged&nbsp;skeletal muscle during disuse and recovery in response to treatment with a secrotome product derived from partially differentiated human pluripotent stem cells.

Aged individuals are at risk to experience slow and incomplete muscle recovery following periods of disuse atrophy. While several therapies have been employed to mitigate muscle mass loss during disuse and improve recovery, few have proven effective at both. Therefore, the purpose of this study was to examine the effectiveness of a uniquely developed secretome product (STEM) on aged skeletal muscle mass and function during disuse and recovery. Aged (22&nbsp;months) male C57BL/6 were divided into PBS or STEM treatment (n = 30). Mice within each treatment were assigned to either ambulatory control (CON; 14&nbsp;days of normal cage ambulation), 14&nbsp;days of hindlimb unloading (HU), or 14&nbsp;days of hindlimb unloading followed by 7&nbsp;days of recovery (recovery). Mice were given an intramuscular delivery into the hindlimb muscle of either PBS or STEM every other day for the duration of their respective treatment group. We found that STEM-treated mice compared to PBS had greater soleus muscle mass, fiber cross-sectional area (CSA), and grip strength during CON and recovery experimental conditions and less muscle atrophy and weakness during HU. Muscle CD68 +,&nbsp;CD11b + and CD163 + macrophages were more abundant in STEM-treated CON mice compared to PBS, while only CD68 + and&nbsp;CD11b + macrophages were more abundant during HU and recovery conditions with STEM treatment. Moreover, STEM-treated mice had lower collagen IV and higher Pax7 + cell content compared to PBS across all experimental conditions. As a follow-up to examine the cell autonomous role of STEM on muscle, C2C12 myotubes were given STEM or horse serum media to examine myotube fusion/size and effects on muscle transcriptional networks. STEM-treated C2C12 myotubes were larger and had a higher fusion index and were related to elevated expression of transcripts associated with extracellular matrix remodeling. Our results demonstrate that STEM is a unique cocktail that possesses potent immunomodulatory and cytoskeletal remodeling properties that may have translational potential to improve skeletal muscle across a variety of conditions&nbsp;that adversely effect aging muscle

Acute Effects of Cheddar Cheese Consumption on Circulating Amino Acids and Human Skeletal Muscle

Cheddar cheese is a protein-dense whole food and high in leucine content. However, no information is known about the acute blood amino acid kinetics and protein anabolic effects in skeletal muscle in healthy adults. Therefore, we conducted a crossover study in which men and women (n = 24; ~27 years, ~23 kg/m2) consumed cheese (20 g protein) or an isonitrogenous amount of milk. Blood and skeletal muscle biopsies were taken before and during the post absorptive period following ingestion. We evaluated circulating essential and non-essential amino acids, insulin, and free fatty acids and examined skeletal muscle anabolism by mTORC1 cellular localization, intracellular signaling, and ribosomal profiling. We found that cheese ingestion had a slower yet more sustained branched-chain amino acid circulation appearance over the postprandial period peaking at ~120 min. Cheese also modestly stimulated mTORC1 signaling and increased membrane localization. Using ribosomal profiling we found that, though both milk and cheese stimulated a muscle anabolic program associated with mTORC1 signaling that was more evident with milk, mTORC1 signaling persisted with cheese while also inducing a lower insulinogenic response. We conclude that Cheddar cheese induced a sustained blood amino acid and moderate muscle mTORC1 response yet had a lower glycemic profile compared to milk