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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 2 Hz 10 Hz Time (min) Kushmerick & al., 1985 Changes in blood composition • Lactate appears ~3 min • pH falls in parallel • Norepinepherine Gaitanos &al 1993 5 min exercise 10 min recovery Mechanical performance changes • P0 declines (atrophy) • Vmax declines (slower) • Endurance increases 2 weeks CLFS Control muscle Jarvis, 1993 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 a2 is more sensitive to AMP • AMP activated protein kinase – Catalytic a subunit – Regulatory b subunit – AMP-binding g subunit • AMPK-kinase Incubate with phosphatase – Liver Kinase B1 (LKB1) – STE-related adaptor (STRAD) a2 is more sensitive to – MOL25 • CaMKK phosphorylation, and has stronger autophos Add phosphatase inhibitor Salt & al., 1998 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) ie: activation of AMPK dis-inhibits FA oxidation, blocks protein translation and activates protein degradation – mTORC1 control of protein translation • FOXO3a, AREBP, HNF4a (activated) – MafBx, autophagy genes AMPK metabolic effects • AICAR treatment – AICARZMP≈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 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 g cofactor 1a • 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 (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 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+ CHO depletion AMP 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 Promoter construct expression combined with knockdown of various NFATs Calabria & al., 2009 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
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