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
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
Glucose and FFA liberation• 70% VO2 max, 2h• Muscle glycogen
falls• Energetic
molecules released from non-muscle stores
Krssak & al 2000
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
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
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
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
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
Mechanical performance changes• P0 declines (atrophy)
• Vmax declines (slower)
• Endurance increases
Jarvis, 1993
Control muscle
2 weeks CLFS
Structural adaptation• Reduced T-tubules• Wider Z-lines• More mitochondria• More capillaries
Eisenberg, 1985
Normal
Stimulated
Stimulation Recovery
Z-li
ne w
idth
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
Endurance adaptation paradigm• Elevated calcium and AMP activate
mitochondrial genes– AMPK, PGC-1, pPAR, MEF2
• Elevated calcium activates muscle genes
Baar, 2006
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
Calcium controls everythinghttp://www.genome.jp/kegg-bin/show_pathway?hsa04020
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
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
NFAT• Stimulation-dependent nuclear translocation
– 30 minutes, 10 Hz; recovery
Liu & al 2001
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)
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
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
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
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
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
CaMK effectors• MEF2• CREB
– CBP/p300 Histone Acetyltransferase partner– Creatine Kinase, SIK (HDAC)
• PGC-1a– Carnitine palmitoyltransferase– Mitochondrial transcription factor A (Tfam)
VEGF• Vascular Endothelial Growth Factor• Angiogenesis
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