Transcript Calcium Signaling

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
– pCrATP
– ATP2 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-CoAmalonyl-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
– 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
FOXO transcription
• 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
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 O2NADP+ + 2 O2– Cell cycle, fibrosis, inflammation
• Xanthine oxidase
– Plasma membrane
– Xanthine+H2O+ O2  Uric acid + H2O2
Targets of ROS
• NF-kB
– H2O2--|SHIP-1--|NEMOIKKNFkB
– Inflammatory
– SOD, BIM, p53, SNARK, NOS, Mt biogenesis
• p21Ras
– 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
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
– Defectsuncontrolled 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