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