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