Calcium 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 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 Glucose and FFA liberation • 70% VO2 max, 2h • Muscle glycogen falls • Energetic molecules released from non-muscle stores Krssak & al 2000 Calcium redistribution • Mitochondrial – Rise ~2x during exercise – Remains elevated > 1 hour • Cytoplasmic – Spikes to 1 uM (diminishing) – Baseline to 300 nM • Metabolite imbalance exceeds exercise period Rest 60 min Ex 30 min Rest 60 min Rest 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 Normal 10 Hz • Stimulation frequency influences – Calcium kinetics – Troponin kinetics – Myosin kinetics 40 Hz Stim frequency contraction time • TA (fast muscle) – No tenotomy no atrophy • Stim effects – Slower – Reduce Twitchtetanus ratio – Reduce sag 10 Hz Twitch forces Tetanic forces Mechanical performance changes • P0 declines (atrophy) • Vmax declines (slower) • Endurance increases 2 weeks CLFS Control muscle Jarvis, 1993 Structural adaptation • • • • Normal Reduced T-tubules Wider Z-lines More mitochondria More capillaries Z-line width Stimulated Stimulation Recovery Eisenberg, 1985 Endurance training • Typically 6 weeks, 5/week 30-120 min @ 6080% VO2max • Performance & oxygen adapts • Contractile proteins less so Heart Rate Lactate Pre-train 6 wks 6 mos Power (watt) 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 everything http://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 CnA CnB • Functional targets – DHPR – BAD CaM Li & al., 2011 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 Activation Domain: HDAC/MRF interactions MEF2 protein map (NLM) 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 Frequent activity Infrequent activity Low Resting Calcium Transient Calcium Spike Cn Inactive High Resting Calcium CaMK Active Myosin Actin HDAC2 Cn Active MEF2 Myoglobin NFAT 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
