calcium signaling describe models of low-force overuse identify the main calcium-dependent signaling...
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Calcium Signaling• Describe models of low-force overuse• Identify the main calcium-dependent signaling
molecules and their mechanism• Explain how calcium homeostasis contributes
to muscle adaptation
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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
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Acute changes during contraction• Phosphate redistribution
– pCrATP– ATP2 Pi + AMP
• pH decline
Kushmerick & al., 1985
2 Hz
10 Hz
Time (min)
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Changes in blood composition• Lactate appears ~3 min• pH falls in parallel• Norepinepherine
5 min exercise 10 min recovery
Gaitanos &al 1993
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Glucose and FFA liberation• 70% VO2 max, 2h• Muscle glycogen
falls• Energetic
molecules released from non-muscle stores
Krssak & al 2000
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Rest 60 min Ex 30 min Rest 60 min Rest
Calcium redistribution• Mitochondrial
– Rise ~2x during exercise– Remains elevated > 1 hour
• Cytoplasmic– Spikes to 1 uM (diminishing)– Baseline to 300 nM
• Metabolite imbalance exceedsexercise period
Madsen & al., 1996
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Stimulation-dependent signaling• Calcium
– Troponin/tropomyosin: contraction– Calcineurin: gene transcription– Calpain: structural remodeling– CaMK: transcription, channel activity
• Energy/ATP– AMP kinase: glucose transport, protein balance– PPAR: mitochondrial hypertrophy– ROS: complicated
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Chronic electrical stimulation• Stanley Salmons & Gerta Vrbova, 1969• Spinal-isolated & tenotomized soleus
– ie: no voluntary or reflex activation– Normally highly active muscle– Stimulate 1-40 Hz, 67% duty cycle 8 hr/day
• Implanted stimulator tibialis anterior– 24/7, 10 Hz– Normally low activity muscle
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Stim frequency contraction time• Soleus (slow muscle)
– Tenotomyatrophy– Tenotomyfaster– Tenotomy+low frequency
preserve speed– Tenotomy+high frequency
faster
• Stimulation frequency influences– Calcium kinetics– Troponin kinetics– Myosin kinetics
Normal
10 Hz
40 Hz
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Stim frequency contraction time• TA (fast muscle)
– No tenotomy no atrophy
• Stim effects– Slower– Reduce Twitch-
tetanus ratio– Reduce sag
10 Hz
Twitch forces Tetanic forces
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Mechanical performance changes• P0 declines (atrophy)
• Vmax declines (slower)
• Endurance increases
Jarvis, 1993
Control muscle
2 weeks CLFS
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Structural adaptation• Reduced T-tubules• Wider Z-lines• More mitochondria• More capillaries
Eisenberg, 1985
Normal
Stimulated
Stimulation Recovery
Z-li
ne w
idth
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Endurance training• Typically 6 weeks, 5/week 30-120 min @ 60-
80% VO2max• Performance & oxygen adapts• Contractile proteins less so
Lact
ate
Hea
rt R
ate
Power (watt)
Pre-train6 wks6 mos
Hoppeler & al 1985
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Endurance adaptation paradigm• Elevated calcium and AMP activate
mitochondrial genes– AMPK, PGC-1, pPAR, MEF2
• Elevated calcium activates muscle genes
Baar, 2006
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Ca mediated protein modification• CaMK (I – IV)
– Calmodulin mediated– Serine/threonine kinases– CaMK-III = eEF2 kinase– Post-synaptic density
• Protein kinase C• Calcineurin
– Calmodulin mediated– Serine/threonine phosphatase
• Calpain (I-III)– Cysteine protease– Cytoskeletal remodeling
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Calcium controls everythinghttp://www.genome.jp/kegg-bin/show_pathway?hsa04020
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Calcineurin (Cn)• Calcium & calmodulin dependent• Serine/threonine phosphatase• High calcium sensitivity: 200 nM• Transcriptional targets
– NFAT– MEF2
• Functional targets– DHPR– BAD
Li & al., 2011
CnBCnA
CaM
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MEF2• MEF2 A/C/D “MADS-box” transcription factor
– Compliment myogenic regulatory factors– Cn and p38-dependent– Blocked by class 2 HDACs– MHC, MLC, Tm, Tn– NADH dehydrogenase (complex 1), GLUT4
MEF2 protein map (NLM)
Activation Domain: HDAC/MRF interactions
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NFAT• Stimulation-dependent nuclear translocation
– 30 minutes, 10 Hz; recovery
Liu & al 2001
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NFAT• NFAT 1/2/3/4 transcription factor
– MEF2, AP-1 cooperation– Cn, GSK3, PKA dependent– Sensitive to mitochondrial calcium handling– Myoglobin, TnI(slow), MHC(slow)
NFAT protein map (NLM)
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SURE and FIRE• Slow Upstream Regulatory Element (SURE)
– Identified in TnI-slow– 110 bp, contains both MEF2, E-box, GT-box
• Fast Intronic Regulatory Element (FIRE)– Identified in TnI-fast– 150 bp in Intron 1, MEF2, E-Box, GT-box
• NFAT-binding– Upstream: promoter– Intron: repressor
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HDAC• Histone deacetylase : gene inactivation• HDAC 2-5; Sirt• MEF2 compliment• CaMK/PDK1 phosphorylation
– Nuclear export– 14-3-3 binding
• ie: blocks MEF2-mediated transcription when not phosphorylated
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Activity dependent transcription
Infrequent activity
Frequent activity
Low Resting Calcium
High Resting Calcium
Transient Calcium Spike
Cn Active
CaMK Active
Cn Inactive
MEF2
NFAT
HDAC2Myosin
Actin
Myoglobin
NADH-D
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CaMKII autophosphorylation• CaM Kinase II (CaMKII)
– CaM dependent kinase– CaM kd = 2 nM, koff 0.3/s
– High affinity, fast kinetics
• Phospho-CaMKII– CaM independent kinase– CaM kd = 0.1 pM, koff 10-6/s
– Very high affinity, slow kinetics
• CaMKII autophosphorylation locks itself in an active conformation
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Rate decoding• Autophosphorylation is like integration• Dephosphorylation is like a high pass filter• eg: Deliver regular calcium pulses
– Measure Ca independent activity– Elevated > 1 hr after exercise in muscle
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CaMK effectors• MEF2• CREB
– CBP/p300 Histone Acetyltransferase partner– Creatine Kinase, SIK (HDAC)
• PGC-1a– Carnitine palmitoyltransferase– Mitochondrial transcription factor A (Tfam)
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VEGF• Vascular Endothelial Growth Factor• Angiogenesis
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Summary• Sustained contractile activity disrupts calcium
and ATP homeostasis• Calcium-dependent kinases (CaMK) and
phosphatasis (Cn) alter transcription (MEF2, NFAT, PGC1)
• Altered gene expression results in mitochondrial biogenesis and calcium buffering
• Subsequent activity causes less disruption