Presentation on cellular causes of muscle weakness after performance of eccentric contractions in mouse skeletal muscle

1. Mechanisms of Strength Loss
Early After Exercise-Induced
Muscle Injury
Christopher P. Ingalls, Ph.D.
Professor
Muscle Biology Laboratory
Department of Kinesiology and Health
Georgia State University
American College of Sports Medicine Annual Meeting
May 28, 2015

2. Unaccustomed Exercise Injures Muscle

3. High Forces and Sarcomere Strain Associated
with Eccentric Contractions Injures Muscle
Concentric

4. Characteristics of Exercise-Induced
Muscle Injury: Membrane Disruption

5. Characteristics of Exercise-Induced
Muscle Injury: Myofibrillar Damage

6. Characteristics of Exercise-Induced
Muscle Injury: Cytoskeletal Disruption

7. Characteristics of Exercise-Induced
Muscle Injury: Triad Disruption

8. Characteristics of Exercise-Induced
Muscle Injury: Strength Deficits
Ingalls et al., J Mus Res Cell Motil. 19: 215, 1998

9. Time Course of Muscle Injury
Days after injury
0 7 14 21 28 35
Strength Deficit
Cell Regeneration
Cell Degeneration
Pain
Cytoskeletal Damage
Membrane Damage
Inflammation
Sarcomere Damage
Edema

10. Exercise-Induced Muscle Injury:
Possible Causes of Strength Deficits
Warren et al., Exerc Sports Sci. Rev. 2: 82, 2001

11. Skeletal Muscle Force Production
SR Z-line
Plasmalemma
SR Z-line
Triad
T-tubule
SR M-line

12. E-C Coupling: SR Ca2+ Release
Ca2+
Ca2+
Ca2+
Ca2+
Ca2+
Ca2+
SERCA
SERCA
RyR1
FKBP12
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+

13. Muscle Force Depends on [Ca2+]i

14. Injury-Induced SR Ca2+ Release Dysfunction:
Voltage-Induced Force vs. Caffeine-Induced Force
Warren et al., J Physiol. 468: 487, 1993
Pre
Post
Caffeine
Pre
Post
Caffeine
Greater relative decreases in voltage-induced force vs.
caffeine-induced force is suggestive of impaired SR Ca2+
release
Isometric Eccentric

15. 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

16. 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

17. 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

18. 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
[3
H]PN200-110
Time after injury (days)
0 3
Bmax(pmol/muscle)
0
2
4
6
8
10
Control
Injured
a
a
bb
DHPR: [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

19. 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
[3
H]ryanodine
Time after injury (days)
0 3
Bmax(pmol/muscle)
0
2
4
6
8
10
Control
Injured
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

20. 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
†
†

21. Possible Causes of Impaired RyR1 Function:
Post-Translational Modifications
Time after injury (days)
Control 3 14
GSH:RyR1
0
1
2
3
4
Time after injury (days)
Control 0 3 14
RelativeS-NitrosylationofRyR1
0.00
0.05
0.10
0.15
0.20
0.25
0.30
*
*
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, 2008Ingalls et al (unpublished observation)

22. 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

23. 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

24. 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

25. 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

26. 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)

27. 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)

28. 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

29. Possible Mechanism of Protein Loss:
Loss of Proteins Due to Membrane Disruption
Ingalls et al (unpublished findings)
JP & isometric
contractions
MWS TA 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

30. Impaired SR Ca2+ Release Model:
Uninjured Muscle
Ca2+
Ca2+
Ca2+
Ca2+
Ca2+
Ca2+
SERCA
SERCA
RyR1
FKBP12
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

31. Ca2+
Ca2+
Ca2+
Ca2+
Ca2+
Ca2+
SERCA
RyR1
FKBP12
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

32. Ca2+
Ca2+
Ca2+
Ca2+
Ca2+
Ca2+
SERCA
RyR1
FKBP12
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

33. 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

34. Acknowledgements
Georgia State University
Cory Baumann Benjamin T. Corona
Russ Rogers Clement Rouviere
Nidhi Gahlot Talal Nofal
Texas A&M University
R.B. Armstrong
Gordon Warren (Georgia State University)
Dawn Lowe (University of Minnesota, Minneapolis)
Baylor College of Medicine
Susan L. Hamilton
National Institutes of Health