1. 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

2. 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

3. Acute changes during contraction Phosphate redistribution – pCr  ATP – ATP  2 Pi + AMP pH decline Kushmerick & al., 1985 2 Hz 10 Hz Time (min)

4. Changes in blood composition Lactate appears ~3 min pH falls in parallel Norepinepherine 5 min exercise10 min recovery Gaitanos &al 1993

5. Glucose and FFA liberation 70% VO2 max, 2h Muscle glycogen falls Energetic molecules released from non-muscle stores Krssak & al 2000

6. Rest60 min Ex30 min Rest60 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 exceeds exercise period Madsen & al., 1996

7. 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

8. 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

9. Stim frequency  contraction time Soleus (slow muscle) – Tenotomy  atrophy – Tenotomy  faster – Tenotomy+low frequency  preserve speed – Tenotomy+high frequency  faster Stimulation frequency influences – Calcium kinetics – Troponin kinetics – Myosin kinetics Normal 10 Hz 40 Hz

10. Stim frequency  contraction time TA (fast muscle) – No tenotomy  no atrophy Stim effects – Slower – Reduce Twitch- tetanus ratio – Reduce sag 10 Hz Twitch forcesTetanic forces

11. Mechanical performance changes P 0 declines (atrophy) V max declines (slower) Endurance increases Jarvis, 1993 Control muscle 2 weeks CLFS

12. Structural adaptation Reduced T-tubules Wider Z-lines More mitochondria More capillaries Eisenberg, 1985 Normal Stimulated StimulationRecovery Z-line width

13. Endurance training Typically 6 weeks, 5/week 30-120 min @ 60- 80% VO2max Performance & oxygen adapts Contractile proteins less so Lactate Heart Rate Power (watt) Pre-train 6 wks 6 mos Hoppeler & al 1985

14. Endurance adaptation paradigm Elevated calcium and AMP activate mitochondrial genes – AMPK, PGC-1, pPAR, MEF2 Elevated calcium activates muscle genes Baar, 2006

15. 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

16. Calcium controls everything http://www.genome.jp/kegg-bin/show_pathway?hsa04020

17. Calcineurin (Cn) Calcium & calmodulin dependent Serine/threonine phosphatase High calcium sensitivity: 200 nM Transcriptional targets – NFAT – MEF2 Functional targets – DHPR – BAD Li & al., 2011 CnB CnA CaM

18. 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

19. NFAT Stimulation-dependent nuclear translocation – 30 minutes, 10 Hz; recovery Liu & al 2001

20. 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)

21. 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

22. 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

23. Activity dependent transcription Infrequent activity Frequent activity Low Resting Calcium High Resting Calcium Transient Calcium Spike Cn Active CaMK Active Cn Inactive MEF2 NFAT HDAC2 Myosin Actin Myoglobin NADH-D

24. CaMKII autophosphorylation CaM Kinase II (CaMKII) – CaM dependent kinase – CaM k d = 2 nM, k off 0.3/s – High affinity, fast kinetics Phospho-CaMKII – CaM independent kinase – CaM k d = 0.1 pM, k off 10 -6 /s – Very high affinity, slow kinetics CaMKII autophosphorylation locks itself in an active conformation

25. 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

26. CaMK effectors MEF2 CREB – CBP/p300 Histone Acetyltransferase partner – Creatine Kinase, SIK (HDAC) PGC-1a – Carnitine palmitoyltransferase – Mitochondrial transcription factor A (Tfam)

27. VEGF Vascular Endothelial Growth Factor Angiogenesis

28. 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