Metabolic Signaling• Describe models of low-force overuse• Identify the main energy-dependent signaling

molecules and their mechanisms– AMPK– PGC-1a– GSK– Reactive oxygen

Low force overuse• Models

– Chronic stimulation– Endurance training

• Physiological stresses– Electrophysiological– Oxygen delivery/handling– ATP metabolism

• Adaptation– SR swelling– Mitochondrial hypertrophy– “Slow” phenotype expression– Atrophy

Acute changes during contraction• Phosphate redistribution

– pCrATP– ATP2 Pi + AMP

• pH decline

Kushmerick & al., 1985

2 Hz

10 Hz

Time (min)

Changes in blood composition• Lactate appears ~3 min• pH falls in parallel• Norepinepherine

5 min exercise 10 min recovery

Gaitanos &al 1993

Mechanical performance changes• P0 declines (atrophy)

• Vmax declines (slower)

• Endurance increases

Jarvis, 1993

Control muscle

2 weeks CLFS

Cellular energy sensors• AMP kinase: glucose transport, protein

balance• PPAR: mitochondrial hypertrophy• GSK: hormonal/systemic integration• ROS: complicated

Endurance adaptation paradigm• Elevated calcium and AMP activate

mitochondrial genes– AMPK, PGC-1, pPAR, MEF2

• Elevated calcium activates muscle genes

Baar, 2006

AMPK• AMP activated protein

kinase– Catalytic subunit– Regulatory subunit– AMP-binding subunit

• AMPK-kinase– Liver Kinase B1 (LKB1)– STE-related adaptor (STRAD)– MOL25

• CaMKKSalt & al., 1998

2 is more sensitive to AMP

2 is more sensitive to phosphorylation, and has stronger autophos

Incubate with phosphatase

Add phosphatase inhibitor

AMPK-Calcium synergy• 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

AMPK analogs• 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

AMPK alters metabolism and growth• Acetyl-coenzyme A carboxylase (ACC, inhibited)

– Ac-CoAmalonyl-CoA– Key enzyme in gluconeogenesis– Malonyl-CoA blocks FA import to mitochondria

• PFK3B (activated)– F1-p F1,6-pp

• TSC2, raptor (inhibited)– mTORC1 control of protein translation

• FOXO3a, AREBP, HNF4a (activated)– MafBx, autophagy genes

ie: activation of AMPK dis-inhibits FA oxidation, blocks protein translation and activates protein degradation

AMPK metabolic effects• AICAR treatment

– AICARZMP≈AMP– 5 days

• Inhibits ACC• Upregulates GLUT4 & HK• LKB1-dependent

Holmes & al., 1999

AMPK activation facilitates glucose uptake, glycolysis, and fatty acid transport. ie: production or replenishment of ATP

FOXO transcription• Counter-regulation by Akt/AMPK

– Autophagy: ATG– Atrophy: MuRF MafBx– Arrest: p21, p27– Apoptosis: BIM, fas– Angiogenesis– Energy: PGC1a, HK

• Insulin/IGFAkt• AMPAMPK

Salih & Brunet, 2008

PGC-1a• Peroxisome proliferator activated receptor

cofactor 1• 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

Fast-muscle specific PGC1 overexpression

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

Lin & al ., 2002

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

GSK3• Glycogen synthase kinase 3 ()

– 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

Reactive oxygen species• Oxygen radical (O2

-, H2O2, OH∙) signaling/damage

Powers & Jackson 2008

Sources of ROS• Electron transport chain

– Electron “leakage” through Complex I,III centers– Cytochrome-C, ubiquinone– Antioxidant expression

• NAD(P)H oxidase– SR/T-tubules– NADPH + 2 O2NADP+ + 2 O2

-

– Cell cycle, fibrosis, inflammation

• Xanthine oxidase– Plasma membrane– Xanthine+H2O+ O2 Uric acid + H2O2

Targets of ROS• NF-kB

– H2O2--|SHIP-1--|NEMOIKKNFkB– Inflammatory– SOD, BIM, p53, SNARK, NOS, Mt biogenesis

• p21Ras – Oxidation of cysteine residues increases GTP exchange– PI-3K, MAPKprotein turnover

• Src– Oxidation of C245 and C487 increases kinase– Myoblast proliferation– AKAP121-enhanced Mt ATP synthesis

Contractile activity

Ca2+ AMP CHO depletion O2-

Cn CaMK PKC AMPK GSK Src IKK Ras

NFAT MEF2 CREB PGC-1 FOXO TSC2 NF-kb Rb

Contractile proteins

Mitochondrial proteins Angiogeneis

Combinatorial control of genes• Multiple elements in promoter-proximal region

– Cooperative: multiple elements combine to recruit transcription complex

– Competitive: overlapping domains block each other

– Nonlinear: transcriptosome

• Intron elements

MHC control• NFAT isoforms• Intergenic antisense• Intronic miRNA

Calabria & al., 2009

Promoter construct expression combined with knockdown of various NFATs

Cancer parallels• Proto oncogenes

– LKB1, PGC-1, p53, etc– Negative controllers of growth– Defectsuncontrolled growth

• Chemotherapy often targets these pathways– Exaggerated muscle loss– Weakness, fatigue

Summary• 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