Exploring the role of BMP7 gene expression in an In Vitro model of aging human skeletal muscle.

Sarcopenia is an age-related condition associated with rapid loss of skeletal muscle (SKM) tissue that affects mobility and quality of life of geriatric individuals. Mechanistic Target of Rapamycin (mTOR) and Protein Kinase B (AKT) have significant roles in SKM hypertrophy with responses to DNA damage and repair within SKM. However, mTOR and AKT expression is significantly decreased with age. Upstream of AKT, Bone Morphogenetic Protein (BMP7) is a member of the TGF-β signaling family that has been reported as a positive regulator of muscle hypertrophy through the Bmp–Smad1/5/8 signaling axis. PURPOSE: To use an in vitro model of aging muscle cells to investigate the role of BMP7 expression on protein synthesis. METHODS: Human SKM myoblasts were cultured and grown beyond mature myotube formation (typically day 6) to emulate aged SKM tissue (extracted on day 18). Groups included control cells (D6) and aged SKM myotubes (D18). Total RNA was extracted at the respective time points (days 6 & 18) and gene expression for BMP7, mTOR, and AKT was determined by qPCR. RESULTS: BMP7 expression was 7.73 fold greater for D18 compared to D6 (p \u3c 0.05). No differences were reported for AKT or mTOR. Data are expressed as fold changes. CONCLUSION: BMP7 expression, thought to be a positive regulator of muscle hypertrophy, was increased in the aging muscle cells of our model, despite our hypothesis that it would be decreased. However, BMP7’s downstream targets related to increased protein synthesis, mTOR and AKT, did not similarly increase from D6 to D18, which is constant with the phenomena of sarcopenia. This leads us to speculate that there may be additional mechanisms related to BMP7 activation and, despite increased signaling, may block protein synthesis at the level of AKT

Myogenic Regulatory Factor Expression is Downregulated Following Formoterol Stimulation in Thyroid Hormone Depleted Skeletal Muscle

In skeletal muscle (SKM), gene expression of transcription factors regulating myogenesis are dependent on Thyroid Hormone (TH) signal transduction. Expression of myogenic regulatory factors may be altered due to dysregulated TH metabolism, which may result in SKM dysfunction and intolerance to exercise in individuals with hypothyroidism. PURPOSE: Implement an in vitro model of hypothyroidism in SKM and determine the response of myogenic regulatory factor expression during several stages of myogenesis following TH depletion. Formoterol, an exercise mimetic, was also used to examine the effects of exercise signaling on myogenesis in TH depleted cells. METHODS: Human SKM myoblasts (n = 6 per group) were cultured and differentiated until mature myotube formation (Day 6). Groups included control cells (CON), TH depleted cells (ThD), and TH depleted cells plus formoterol stimulation (ThD+F; 30nm for 3h). Total RNA was extracted during mid-myogenesis (Day 4) and at terminal differentiation (Day 6). Gene expression for myogenic regulatory factors (Myf5, MyoD, MyoG) was determined by qPCR. RESULTS: ThD decreased Myf5 at both Day 4 and Day 6 compared to control (P\u3c0.001). Myf5 was increased following ThD + F compared to ThD at Day 4 (P\u3c0.05). MyoD decreased following ThD at both Day 4 and Day 6 (P\u3c0.001). Further, MyoD was decreased following ThD + F at both Day 4 and Day 6 compared to ThD (P\u3c0.001). ThD had no effect on MyoG at Day 4 and Day 6; however, MyoG was decreased following ThD + F compared to ThD and control at both time points (P\u3c0.001). Data are expressed as mean ± SEM. CONCLUSION: TH depletion had no effect on MyoG but did reduce the expression of both Myf5 and MyoD at both Day 4 and Day 6. Additionally, ThD+F resulted in the lowest expression of MyoG and MyoD for both time points. These results indicate TH depletion and formoterol stimulation may inhibit myotube maturation

Mitochondrial Biogenesis is Dysregulated in Thyroid Hormone Depleted Muscle Cells Despite Stimulatory Effects of Formoterol

Skeletal muscle (SKM) is an important regulator of metabolism and adaptations from exercise training influences mitochondrial function. Thyroid hormone (TH) is a regulator of SKM processes, including mitochondrial biogenesis. PURPOSE: To use an in vitro model of hypothyroidism to test the hypothesis that SKM cells will have dysregulated mitochondrial homeostasis. Additionally, the exercise mimetic, formoterol, was used to determine the effects of exercise signaling on mitochondrial biogenesis. METHODS: Human SKM myoblasts (n = 6 per group) were cultured and differentiated until mature myotube formation (Day 6). Groups included control cells (CON), TH depleted cells (ThD), and TH depleted cells plus formoterol stimulation (ThD+F; 30nM for 3h). Total RNA was extracted during mid-myogenesis (Day 4) and at terminal differentiation (Day 6). Gene expression for Peroxisome Proliferator-Activated Receptor Gamma Coactivator-1 Alpha (PGC-1α), Mitochondrial Transcription Factor A (TFAM), and Nuclear Respiratory Factor 1 (NRF1) was determined by qPCR. Data was analyzed by repeated measures ANOVA. RESULTS: PGC-1α: D4 ThD was decreased compared to D4 ThD+F (-4.6); D4 ThD+F was increased compared to D4 CON (4.6); D6 CON was decreased compared to D6 ThD+F (-2.9); D6 ThD was decreased compared to D6 ThD+F (-3.7). TFAM: D4 ThD+F was greater than D4 CON (3.6); D4 ThD+F was greater than D6 ThD+F (3.6); D6 ThD was decreased compared to D6 CON (-0.55); D6 ThD+F was decreased compared to D6 CON (-0.63). NRF1: D4 ThD was decreased compared to D4 CON (-0.31); D4 ThD was greater than D4 ThD+F (0.36); D4 ThD was greater than D6 ThD (0.17); ThD+F was decreased compared to D4 CON (-0.67); D6 CON was decreased compared to D4 CON (-0.18); D6 ThD was decreased compared to D6 CON (-0.3); D6 ThD+F was decreased compared to D6 CON (-0.42). All reported differences are significant (p \u3c 0.05). Data are expressed as fold changes. CONCLUSION: ThD media resulted in reduced NRF1 signaling in both D4 and D6 with a subsequent decrease in D6 only for TFAM. Formoterol resulted in the expected stimulation of PGC-1α at both D4 and D6, but subsequent signaling for genes associated with mitochondrial biogenesis common to PGC-1α stimulation were lost as a result of TH depletion at D6 only for TFAM and both D4 and D6 for NRF1

Formoterol Stimulation In Vitro Influences Myogenic Regulatory Factors During Myogenesis in Human Skeletal Muscle Cells

The process of myogenesis within skeletal muscle (SKM) is essential for growth and repair and is coordinated via the expression of myogenic regulatory genes. Previous animal studies have reported that formoterol, a beta-adrenergic receptor agonist, has stimulating effects on genes related to SKM mitochondrial function and biogenesis, similar to effects found for exercise. Lesser known is the potential “exercise mimetic” influence that formoterol stimulation may have during the stages of myogenesis, especially in human SKM cells. PURPOSE: To investigate the effects of formoterol stimulation on expression of myogenic regulatory genes during myogenesis in human SKM cells. METHODS: Human SKM myoblasts (n = 6 per group) were cultured and differentiated until mature myotube formation (Day 6). Groups included control cells (CON) and cells stimulated by 30nM formoterol for 3h prior to RNA extraction points (FORM). Total RNA was extracted during mid-myogenesis (Day 4) and at terminal differentiation (Day 6) (a cell culture model of investigating myogenesis). Gene expression for Myogenic factor 5 (Myf5), Myogenic differentiation 1(MyoD), and Myogenin (MyoG) was determined by qPCR. Data was analyzed using repeated measures ANOVA. RESULTS: Myf5: There was no change for either condition for D4. D6 CON was lower than D4 CON (-0.25). D6 FORM was greater than D4 FORM (0.65) and D6 CON (0.75). MyoD: D4 FORM was lower than D4 CON (-0.57). D6 FORM was greater than D4 FORM (0.85) and lower than D6 CON (-0.16). D6 CON was lower than D4 CON (-0.33). MyoG: D4 FORM was lower than D4 CON (-0.72). D6 CON was lower than D4 CON (-0.44). D6 FORM was lower than D6 CON (-0.24). All reported differences are significant (p \u3c 0.05). Data are expressed as fold changes. CONCLUSION: As expected, for the CON group, Myf5, MyoD, and MyoG expression all decreased from D4 mid-myogenesis to D6 terminal myogenesis, indicating finalization of the myogenic gene program. For the FORM group, Myf5 expression was elevated at D6 compared to CON while MyoG and MyoD expression was lower than CON for D4 and D6. The interpretation is that FORM stimulation increased stimulus of D4 myoblast proliferation and, thus, delayed initiation of differentiation. These results, coupled with other preliminary data from our lab showing increased mitochondrial biogenesis with this model of investigation, suggests that this exercise mimetic stimulation may cause shift in the cell towards bioenergetic preference rather than fusion of myotubes