• Describe models of low-force overuse
• Identify the main energy-dependent signaling molecules and their mechanisms
– AMPK
– PGC-1a
– GSK
– Reactive oxygen

• Models
– Chronic stimulation
– Endurance training
• Physiological stresses
– Electrophysiological
– Oxygen delivery/handling
– ATP metabolism
• Adaptation
– SR swelling
– Mitochondrial hypertrophy
– “Slow” phenotype expression
– Atrophy

• Phosphate redistribution
– pCr  ATP
– ATP  2 Pi + AMP
• pH decline
Kushmerick & al., 1985
Time (min)
2 Hz
10 Hz

5 min exercise 10 min recovery
• Lactate appears ~3 min
• pH falls in parallel
• Norepinepherine
Gaitanos &al 1993

• P
0 declines (atrophy)
• V max declines (slower)
• Endurance increases
2 weeks CLFS
Control muscle
Jarvis, 1993

• AMP kinase: glucose transport, protein balance
• PPAR: mitochondrial hypertrophy
• GSK: hormonal/systemic integration
• ROS: complicated

• Elevated calcium and AMP activate mitochondrial genes
– AMPK, PGC-1, pPAR, MEF2
• Elevated calcium activates muscle genes
Baar, 2006

• AMP activated protein kinase
– Catalytic a subunit
– Regulatory b subunit
– AMP-binding g subunit
• AMPK-kinase
•
– Liver Kinase B1 (LKB1)
– STE-related adaptor
(STRAD)
– MOL25
CaMKK a
2 is more sensitive to phosphorylation, and has stronger autophos a
2 is more sensitive to AMP
Incubate with phosphatase
Add phosphatase inhibitor
Salt & al., 1998

• CaMKK activates AMPK only in the presence of
AMP
– AMP protects from phosphatase activity (PP2c)
– CAMKK, but not LKB1 activated by exercise
– Starvation vs activity

• LKB1-STRAD-MOL25 substrates
– Tumor suppressor, esp smooth muscle
– HeLa cells are LKB1-/-
• SNARK
– Required for exercise-stimulated glucose uptake
– Blocked in insulin-resistant
• MARK1-4
LKB1 ko reduces activation of SNARK by exercise
SNARK ko reduces activation of GLUT4 by exercise
Koh & al 2012

• Acetyl-coenzyme A carboxylase (ACC, inhibited)
– Ac-CoA  malonyl-CoA
– Key enzyme in gluconeogenesis
– Malonyl-CoA blocks FA import to mitochondria
• PFK3B (activated)
– F1-p  F1,6-pp ie: activation of AMPK dis-inhibits
FA oxidation, blocks protein translation and activates protein degradation • TSC2, raptor (inhibited)
– mTORC1 control of protein translation
• FOXO3a, AREBP, HNF4a (activated)
– MafBx, autophagy genes

• AICAR treatment
– AICAR  ZMP≈AMP
– 5 days
• Inhibits ACC
• Upregulates GLUT4 & HK
• LKB1-dependent
AMPK activation facilitates glucose uptake, glycolysis, and fatty acid transport. ie: production or replenishment of ATP
Holmes & al., 1999

• Counter-regulation by Akt/AMPK
– Autophagy: ATG
– Atrophy: MuRF MafBx
– Arrest: p21, p27
– Apoptosis: BIM, fas
– Angiogenesis
– Energy: PGC1a, HK
• Insulin/IGF  Akt
• AMP  AMPK
Salih & Brunet, 2008

• Peroxisome proliferator activated receptor g cofactor 1 a
• Broad spectrum coordinator of nuclear and mitochondrial transcription
– Antioxidant enzymyes: SOD, catalase, GPx1, UCP
– Inflammatory response: TNF-a, IL-6 (down)
– Mt biogenesis: Tfam, Cytochrome oxidase
• Co-factor
– MEF2, NFAT, NRF-1

• PGC1 under MCK promoter
• Tg muscles: more mt, COX, myoglobin
• Tg more MHC-1, but still 90% MHC-2
Lin & al ., 2002

• PGC-1a1: mitochondrial biogenesis, oxphos
• PGC-1a4: IGF-1, myostatin repression

• Glycogen synthase kinase 3 ( a,b
)
– Inhibited by phosphorylation: PKB, p38, RSK
– Targets mostly primed substrates
• Inhibits glycogen synthase
• Cell growth control
– C-Myc, Bcl2, MDM2, retinoblastoma (Rb)
– Wnt, NFAT, CREB

• Oxygen radical (O
2
, H
2
O
2
, OH ∙ ) signaling/damage
Powers & Jackson 2008

• Electron transport chain
– Electron “leakage” through Complex I,III centers
– Cytochrome-C, ubiquinone
– Antioxidant expression
• NAD(P)H oxidase
– SR/T-tubules
– NADPH + 2 O
2
 NADP + + 2 O
2
-
– Cell cycle, fibrosis, inflammation
• Xanthine oxidase
– Plasma membrane
– Xanthine+H
2
O+ O
2
 Uric acid + H
2
O
2

• NF-kB
– H2O2--|SHIP-1--|NEMO  IKK  NFkB
– Inflammatory
– SOD, BIM, p53, SNARK, NOS, Mt biogenesis
• p21 Ras
– Oxidation of cysteine residues increases GTP exchange
– PI-3K, MAPK  protein turnover
• Src
– Oxidation of C245 and C487 increases kinase
– Myoblast proliferation
– AKAP121-enhanced Mt ATP synthesis

Contractile activity
Cn CaMK
Ca2+
PKC
AMP
AMPK GSK
CHO depletion
Src IKK
O2-
Ras
NFAT MEF2 CREB PGC-1 FOXO TSC2 NF-kb Rb
Contractile proteins
Mitochondrial proteins
Angiogeneis

• Multiple elements in promoter-proximal region
– Cooperative: multiple elements combine to recruit transcription complex
– Competitive: overlapping domains block each other
– Nonlinear: transcriptosome
• Intron elements

• NFAT isoforms
• Intergenic antisense
• Intronic miRNA
Promoter construct expression combined with knockdown of various NFATs
Calabria & al., 2009

• Proto oncogenes
– LKB1, PGC-1, p53, etc
– Negative controllers of growth
– Defects  uncontrolled growth
• Chemotherapy often targets these pathways
– Exaggerated muscle loss
– Weakness, fatigue

• Prolonged muscle activity stimulates
– Persistently elevated calcium
– ATP stress
– Reactive oxygen stress
• Immediate consequences
– Increased Ox-phos, FA, and glucose uptake
– Suppressed calcium release
• Long-term consequences
– Mitochondrial biogenesis
– Contractile protein isoform switching