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Mechanisms of Strength Loss Early After Exercise-Induced
Muscle Injury
Christopher P. Ingalls, Ph.D.Professor
Muscle Biology LaboratoryDepartment of Kinesiology and Health
Georgia State University
American College of Sports Medicine Annual MeetingMay 28, 2015
Unaccustomed Exercise Injures Muscle
High Forces and Sarcomere Strain Associated with Eccentric Contractions Injures Muscle
Concentric
Characteristics of Exercise-Induced Muscle Injury: Membrane Disruption
Characteristics of Exercise-Induced Muscle Injury: Myofibrillar Damage
Characteristics of Exercise-Induced Muscle Injury: Cytoskeletal Disruption
Characteristics of Exercise-Induced Muscle Injury: Triad Disruption
Characteristics of Exercise-Induced Muscle Injury: Strength Deficits
Ingalls et al., J Mus Res Cell Motil. 19: 215, 1998
Time Course of Muscle Injury
Days after injury
0 7 14 21 28 35Strength Deficit
Cell Regeneration
Cell Degeneration
Pain
Cytoskeletal Damage
Membrane Damage
Inflammation
Sarcomere Damage
Edema
Exercise-Induced Muscle Injury:Possible Causes of Strength Deficits
Warren et al., Exerc Sports Sci. Rev. 2: 82, 2001
Skeletal Muscle Force Production
SR Z-line
Plasmalemma
SR Z-line
Triad
T-tubule
SR M-line
E-C Coupling: SR Ca2+ Release
Ca2+Ca2+
Ca2+
Ca2+Ca2+
Ca2+
SERCA
SERCA
RyR1FKBP12
DHPR
JP1/2
Ca2+Ca2+
Ca2+
CSQ
T-Tubule
Sarcoplasmic Reticulum
Ca2+
Ca2+
Ca2+
CaM
NO
Ca2+
GSHMg ATP P
Ca2+
Ca2+
Ca2+
CaM
NO
Ca2+
GSHMg ATP P
CaM
NO
Ca2+
GSH Mg ATPP
CaM
NO
Ca2+
GSH Mg ATPP
Ca2+ Ca2+ Ca2+ Ca2+Ca2+Ca2+ Ca2+ Ca2+ Ca2+ Ca2+Ca2+Ca2+ Ca2+ Ca2+ Ca2+ Ca2+Ca2+Ca2+
Muscle Force Depends on [Ca2+]i
Injury-Induced SR Ca2+ Release Dysfunction:Voltage-Induced Force vs. Caffeine-Induced Force
Warren et al., J Physiol. 468: 487, 1993
PrePost
Caffeine
Pre
Post
Caffeine
Greater relative decreases in voltage-induced force vs. caffeine-induced force is suggestive of impaired SR Ca2+ release
Isometric Eccentric
Injury-Induced SR Ca2+ Release Dysfunction: Single Muscle Fibers
Balnave & Allen, J Physiol. 488:25,1995
SR Ca2+ transients are reduced in single muscle fibers 10 minutes after eccentric contractions performed ex vivo
Injury-Induced SR Ca2+ Release Dysfunction:EDL Muscle
Ingalls et al., J Appl Physiol. 85: 58, 1998
Immediately after injury 3 days after injury
Time (s)
Voltage-induced SR Ca2+ transients are reduced in muscle fibers immediately and 3 days after eccentric contractions performed in vivo
Possible Causes of Impaired SR Ca2+ Release• Plasma membrane electrophysiology
– Warren et al. J Physiol. 515: 609, 1999– Call et al. J Physiol. 591: 3765, 2014
• T-Tubule membrane integrity– Warren et al. Cell Tissue Res 282: 311, 1995– Yeung et al. J. Physiol. 540: 581, 2002
• DHPR protein (T-Tubule) function and content– Ingalls et al. J Appl Physiol. 85: 58, 1998– Ingalls et al. J. Appl Physiol. 96: 1619, 2004
• RyR1 protein (SR) function, content, and modifications– Function (Ingalls et al. J Appl Physiol. 85: 58, 1998)
– Content (Ingalls et al. J Appl Physiol. 96: 1619, 2004)
– Post-translational modifications (Corona et al. J Appl Physiol. 108: 1542, 2008)
– Loss of FKBP12 (Baumann et al. Physiol Report 2(7): pii e12081, 2014)
• DHPR (T-Tubule) interactions with RyR1 (SR): JP Proteins– Ingalls et al J Appl Physiol. 85: 58, 1998 – Corona et al Am J Physiol: Cell 298: C365, 2010
DHPR Protein Content and Function
DHPR protein content is increased immediately and 3 days after eccentric contractions
Ingalls et al., J. Appl. Physiol. 96: 1619, 2004
[3H]PN200-110
Time after injury (days)0 3
Bm
ax (p
mol
/mus
cle)
0
2
4
6
8
10ControlInjured
aa
bbDHPR: [3H]PN200-110
K+ contracture experiments indicate the function of the DHPR is normal immediately after eccentric contractions (Ingalls et al J Appl Physiol. 85: 58, 1998)
Loss or disruptions in the DHPR protein would not appear to contribute to impaired SR Ca2+ release and muscle weakness during the first 3 days after injury
RyR1 Protein Content
Ingalls et al., J. Appl. Physiol. 96: 1619, 2004
RyR1 protein content is not altered immediately after eccentric contractions but is significantly reduced 3 days after injury
Cav1.1 is increased immediately and 3 days after eccentric contractions
[3H]ryanodine
Time after injury (days)0 3
Bm
ax (p
mol
/mus
cle)
0
2
4
6
8
10ControlInjured
aaa
b
RyR1: [3H]ryanodine
Loss of RyR1 protein would not appear to contribute to impaired SR Ca2+ release and muscle weakness immediately after injury but would 3 days after injury
RyR1 Protein Function
Ingalls et al., J Appl Physiol. 85: 58, 1998
Maximal SR Ca2+ release rate is reduced 6% immediately after eccentric contractions based on Ca2+ mini-electrode measurements
Maximal SR Ca2+ release rates are reduced approximately 20-24% at 3 and 5 days after eccentric contractions based on fluorometric measurements
Gradual reductions in SR Ca2+ release rates in the days after injury would contribute to impaired voltage-induced SR Ca2+ release and muscle weakness
†
†
Possible Causes of Impaired RyR1 Function:Post-Translational Modifications
Time after injury (days)Control 3 14
GS
H:R
yR1
0
1
2
3
4
Time after injury (days)Control 0 3 14
Rel
ativ
e S
-Nitr
osyl
atio
n of
RyR
1
0.00
0.05
0.10
0.15
0.20
0.25
0.30
**
Blocking NO production via L-NAME exacerbates strength loss immediately after eccentric contractions (Corona et al. Muscle Nerve 47: 271, 2013)
Oxidative stress of the RyR1 does not appear to influence RyR1 function after eccentric exercise but reductions in RyR1 S-Nitrosylation may influence SR function in the days after injury
Corona et al., J. Appl. Physiol. 105: 1542, 2008 Ingalls et al (unpublished observation)
S-Nitrosylation of RyR1 is reduced at 3 and 14 days after eccentric contractions, but S-Glutathionylation of RyR1 is unaffected after injury
Possible Causes of Impaired RyR1 Function:Loss of FKBP12
Baumann et al., Physiol Report 2(7): pii e12081, 2014
FKBP12 content is reduced immediately after eccentric contractions and is restored by 3 days, and its cytosolic content is increased at 3 days after injury
Cytosolic fractionPellet fraction
Decreases in FKBP12 content immediately after injury are correlated with muscle strength deficits and increases in resting tension, but alterations at later time points do not appear to affect peak isometric force
DHPR-RyR1 Protein Interaction: JP Knockdown
Decreasing junctophilin 1 and 2, proteins that maintain the apposition of the T-Tubule and SR membranes (and thus DHPR and RyR1 interaction) disrupts triad formation, impairs voltage-induced SR Ca2+ release, and decreases force production (Hirata et al. Biophys J. 90: 4418, 2006; Ito et al. J Cell Biol.154: 1059, 2001 )
Garbino et al., Physiol Genomics, 2009
Ito et al., J Cell Biol. 2001
DHPR-RyR1 Protein Interaction:Loss of Junctophilin Proteins After 1 Injury Bout
Corona et al., Am J Physiol: Cell 298: C365, 2010
A single bout of concentric contractions does not affect muscle strength nor JP protein content
A single bout of eccentric contractions results in immediate and prolonged loss in JP 1 & 2 proteins that coincides with skeletal muscle strength deficits
Corona et al., Am J Physiol: Cell 298: C365, 2010
DHPR-RyR1 Protein Interaction:Loss of Junctophilin Proteins After 2 Injury Bouts
Changes in JP protein (JP1) content are correlated to changes in muscle strength, and appear to contribute to impaired voltage-induced SR Ca2+ release after injury
A second bout of eccentric contractions results in immediate decreases in muscle strength and JP1 protein, and both strength and JP1 are recovered by 3 days after injury
Possible Mechanism of Protein Loss:Increase in [Ca2+ ]i and Calpain Proteolysis
Ingalls et al., J. Appl. Physiol. 85: 58,1998
0 h
3 h
6 h
Significant increases in cytosolic free Ca2+ levels occur during the first 6 hours after eccentric contractions (Ingalls et al 1998) and at 48 h after injury (Lynch et al Cell Calcium 22: 373, 1997)
Early increases in cytosolic free Ca2+ stem from entry from the extracellular space (Warren et al Am J Physiol Reg 282: R1122, 2001) via stretch activated channels (e.g., Zhang et al J Appl Physiol 105: 352, 2008; J Appl Physiol 112: 2077, 2012) and/or from SR Ca2+ channel leak (Bellinger et al PNAS 105: 2198, 2008)
Possible Mechanism of Protein Loss:Increase in [Ca2+ ]i and Calpain Proteolysis
Zhang et al., J. Appl. Physiol. 112: 2077, 2012
µ-Calpain appears to be activated 30 min after eccentric contractions (e.g., Zhang et al 2012) and SR proteins are known calpain substrates (Berchtold et al Physiol Rev 80: 1215, 2000)
Elevated cytosolic free Ca2+ and µ-Calpains can disrupt E-C coupling in single muscle fibers (Verburg et al Am J Physiol Cell 296: C1115, 2009)
Possible Mechanism of Protein Loss:Increase in [Ca2+ ]i and Calpain Proteolysis
Zhang et al., J. Appl. Physiol. 112: 2077, 2012
Blocking Ca2+ entry had no effect on force deficits immediately after eccentric contractions, and only small effects 30 min after eccentric contractions
Consistent with our observations that manipulating extracellular Ca2+, blocking Ca2+ entry, and inhibiting calpain protease activity does not affect strength deficits after eccentric contractions in our mouse injury model (Lowe et al. J Appl Physiol 76: 1445, 1994; Warren et al. Am J Physiol Reg. 282: R1122, 2002; Corona et al. Am J Physiol Cell 298: C365, 2010 )
Calpain proteolysis does not appear to affect SR Ca2+ release and strength loss immediately after eccentric contractions in our injury model, however it may influence these factors at later time points
Possible Mechanism of Protein Loss:Loss of Proteins Due to Membrane Disruption
Ingalls et al (unpublished findings)
JP & isometric contractions
MWSTA KB Bath
JP & eccentric contractions
MWS TAKB
Bath
Performance of eccentric contractions ex vivo results in an immediate loss of JPs to the bathing medium
Disruptions in the plasma membrane allow for a number muscle proteins (e.g., CK, LDH) to be lost from the cell during the performance of eccentric contractions
The mechanism of the loss of JP and FKBP12 immediately after eccentric contractions awaits further study
Impaired SR Ca2+ Release Model:Uninjured Muscle
Ca2+Ca2+
Ca2+
Ca2+Ca2+
Ca2+
SERCA
SERCA
RyR1FKBP12
DHPR
JP1/2
Ca2+Ca2+
Ca2+
CSQ
T-Tubule
Sarcoplasmic Reticulum
Ca2+
Ca2+
Ca2+
CaM
NO
Ca2+
GSHMg ATP P
Ca2+
Ca2+
Ca2+
CaM
NO
Ca2+
GSHMg ATP P
CaM
NO
Ca2+
GSH Mg ATPP
CaM
NO
Ca2+
GSH Mg ATPP
Ca2+Ca2+
Ca2+
Ca2+Ca2+
Ca2+
SERCA
RyR1FKBP12
DHPR
JP1/2
Ca2+Ca2+
Ca2+
CSQ
T-Tubule
Sarcoplasmic Reticulum
CaM
NO
Ca2+
GSH Mg ATPP
CaM
NO
Ca2+
GSH Mg ATPP
Ca2+
Ca2+
Ca2+
CaM
NO
Ca2+
GSHMg ATP P
Ca2+
Ca2+
Ca2+
CaM
NO
Ca2+
GSHMg ATP P
Impaired SR Ca2+ Release Model:Immediately After Eccentric Contractions
Ca2+Ca2+
Ca2+
Ca2+Ca2+
Ca2+
SERCA
RyR1FKBP12
DHPR
JP1/2
Ca2+Ca2+
Ca2+
CSQ
T-Tubule
Sarcoplasmic Reticulum
CaM
NO
Ca2+
GSH Mg ATPP
Ca2+
Ca2+
Ca2+
CaM
GSHMg ATP P
Ca2+
Ca2+
Ca2+
Impaired SR Ca2+ Release Model:3 Days After Eccentric Contractions
Ca2+
CaM
NO
Ca2+
GSH Mg ATPP
Take Home Message
The performance of eccentric contractions disrupts certain triad proteins associated with excitation-contraction coupling which appear to contribute to impaired SR Ca2+ release and skeletal muscle weakness in the days after exercise
AcknowledgementsGeorgia State University
Cory Baumann Benjamin T. Corona Russ Rogers Clement Rouviere Nidhi Gahlot Talal Nofal
Texas A&M UniversityR.B. ArmstrongGordon Warren (Georgia State University)Dawn Lowe (University of Minnesota, Minneapolis)
Baylor College of MedicineSusan L. Hamilton
National Institutes of Health