2. O-GlcNAc signaling
• Novel signaling pathway that
plays a critical role in many
diseases
• Has links to major
biochemical pathways
3. Satellite Cells
• Located under the basal
lamina, close to capillary
• Precursors to skeletal
muscle cells
• Have active and
quiescent states,
quiescent actively
maintained
• Involved in growth and
regeneration following
injury or disease Source: Seeley, Stevens and Tate. Essentials of
Anatomy and Physiology. McGraw-Hill, 2009.
4. Hypothesis
1.O-GlcNAc signaling is necessary in
the maintenance of porcine SC
homeostasis
2.O-GlcNAc signaling modulates
skeletal muscle growth
3.O-GlcNAc signaling participates in
skeletal muscle diseases
11. Function of SC
• Cardiotoxin induced muscle damage
and regeneration in 4 week-old pig
• Inject adenoviral with India Ink
together with Ad-GFP/Ad-OGT; Ad-
scramble shRNA/Ad-OGT shRNA 24h after
damage
• Track the GFP+ve fibers and GFP+ve SCs 14d
post injury
12. Hypothesis
1.O-GlcNAc signaling is necessary in
the maintenance of porcine SC
homeostasis
2.O-GlcNAc signaling modulates
skeletal muscle growth
3.O-GlcNAc signaling participates in
skeletal muscle diseases
13. O-GlcNAc signaling in muscle growth
• Inject adenoviral particles into 2-wk-old-pig
semitendinosus muscle with India Ink
–Ad-GFP
–Ad-OGT
–Ad-scramble shRNA
–Ad-OGT shRNA
• Track the GFP+ve fibers: size, fiber type (I.
Iia, and IIb); metabolism (mitochondria #
and activity- NADH, SDH staining)
14. Hypothesis
1.O-GlcNAc signaling is necessary in
the maintenance of porcine SC
homeostasis
2.O-GlcNAc signaling modulates
skeletal muscle growth
3.O-GlcNAc signaling participates in
skeletal muscle diseases
15. O-GlcNAc signaling in muscle atrophy
via starvation and denervation
• Inject adenoviral particles into 2-wk-old-pig
semitendinosus muscle with India Ink
–Ad-GFP
–Ad-OGT
–Ad-scramble shRNA
–Ad-OGT shRNA
• Track the GFP+ve fibers: size, fiber type (I.
Iia, and IIb); metabolism (mitochondria #
and activity- NADH, SDH staining)
O-linked beta-D-N-acetylglucosamine (O-GlcNAc), a sugar derivative that links carbohydrate, lipid, protein, energy, and nucleotide metabolisms, is a novel nutrient sensor that functions in gluconeogenesis, circadian clock, and insulin resistance
Studies of knockout O-GlcNAc transferase in mice shows O-GlcNAc signaling plays critical role in the maintenance of satellite cell quiescence and function; O-GlcNAc signaling is an important regulator of skeletal muscle contractility and metabolism; O-GlcNAc signaling serves as nutrient sensor in perturbed nutritional status to maintain metabolic homeostasis in skeletal muscle
Differentiate and fuse to augment fibers
Activated cells initially proliferate as skeletal myoblasts before undergoing myogenic differentiation
Greater activation also results in increased expression of myogenic basic helix-loop-helix transcription factors MyoD, myogenin, and MRF4 - all responsible for the induction of myocyte-specific genes. HGF testing is also used to identify active satellite cells. Activated satellite cells also begin expressing muscle-specific filament proteins such as desmin as they differentiate.
Fuse with damaged myotubes and undergo further differentiation and maturation
Between basal lamina and plasma membrane
Activation/ self-renewal vs differentiation comes from niche/environmental cues,
i.e. short term calorie restriction increases satelite cell frequenct and proliferation, improves muscle generation and influences efficiency of engraftment,
i.e. resident stem cell maitenece critically regulated by metabolic signals of niches
Porcine skeletal muscle embryogenesis, postnatal growth, and muscle fiber composition and metabolism are tightly regulated by nutrient flux related to satellite cells because how can they sense environment and know when to grow?
?????????
wound healing as the pig skin closely resembles human skin in innervation, barrier properties and hair follicle distribution
Capable of bringing together major cellular metabolic pathways
New area
Ag: fatty acid oxidation, glucose homeostasis, protein syhnthesis and degradation to lead to branch studies to more efficiently use nutrients to maximize growht, production, and quality of the meat
TheraputicL obesity, muscle atrophy, cancer signaling pathways
Standard identifiers work ihc staining
Pax 7-satellite cell
myoD-myogenic progenior cell
Muscle cell-desmin
Is porcine LD in newborn pig, B shows wihin satellite cells, muscle specific markers and MyoD once activated. If quaiescent, no myoD seen, shows Ab works on pigs,
Gives platform for us to jump off of
We treated porcine SCs with OGT inhibitor TT04 to attenuate O-GlcNAc signaling and OGA inhibitor ThG to accumulate O-GlcNAc signaling to assess the effect of this nutrient sensor pathway on SC proliferation and differentiation. We used BrdU incorporation assay to evaluate the rate of SC proliferation, and found that attenuation of O-GlcNAc signaling impairs SC proliferation (Fig. 3A). In contrast, inhibition of this signaling pathway enhances myogenesis as indicated by increased myosin heavy chain staining and fusion (Fig. 3B). These findings demonstrate that O-GlcNAc signaling play a critical role during porcine skeletal myogenesis.
Though there was contrasting results
BrdU thiamin analog base, reflects proliferation, stain with antibody
Loss of Ogt resulted in reduced SC expansion and self-renewal., by tamoicifen injection, results determined by in vivo regeneration
OGT tamoxifen cuts out the gene from genomic DNA deleted
Why more nuclei on cKO?
Conditional Ogt ablation impaired SC-mediated muscle regeneration. Tibialis anterior (TA) muscle was injected by cardiotoxin (CTX), 7d and later, muscles were harvested
