Extensive mononuclear infiltration and

myogenesis characterize recovery of

dysferlin-null skeletal muscle from contraction-
induced injuries

Joseph A. Roche (1), Richard M. Lovering (1), Renuka Roche (2), Lisa W. Ru (1),
Patrick W. Reed (2), and Robert J. Bloch (1)
1. Department of Physiology, University of Maryland School of Medicine, Baltimore, Maryland
2. Department of Physical Therapy and Rehabilitation Science, University of Maryland School
of Medicine, Baltimore, Maryland
Purposes
• Determine the role of dysferlin in the recovery of skeletal muscle to:
• A few large consecutive injuries
• Many small injuries
• Assess the role of myogenesis in muscle recovery
Why Dysferlin? Dysferlinopathies
• Dysferlin: a protein
• Dysferlinopathies: Mutation of the dysferlin gene
• Phenotype: human muscle diseases characterized by deficits in muscle repair
• Pathogenesis also includes an inflammatory response due to either:
• Necrosis of myofibers after failed membrane repair, or
• Normal macrophage expression

Vernengo, L (2013)
Skeletal muscle injury
• Injury is used a model to gain insights into muscle recovery
• Induced by lengthening contraction
• Disrupts plasma membrane of skeletal muscle
• Used to study the mechanisms by which muscles regain function
• Many small injuries
• Recovery process: repair of sarcolemmal membrane of injured myofibers
• One large injury
• Recovery process: myogenesis without significant sarcolemmal repair
Dysferlin in muscle recovery – previous
study
(Lovering, et a. 2004)

• Induced a single large strain due to lengthening contraction

• Tibialis anterior muscles in rats
• Dysferlin-null mice models recovery to a single large strain:
• Infiltration of mononuclear cells
• Necrotic death of myofibers
• Extensive myogenesis
Hypotheses
• Myogenesis is not necessary for the recovery from a few large injuries
when dysferlin is present, BUT that myogenesis is necessary when the
injuries occur without dysferlin

• Myogenesis is necessary for the recovery from many small injuries

with or without the presence of dysferlin
Injury models and experimental
setup
• Mice ankle dorsiflexors (DF) were the target muscle group
• Groups
• A/WySnJ: expressed dysferlin
• A/J: did not express dysferlin
DYSFERLIN + + - -

IRRADIATTION - + - +

• Small injuries
• Ankle plantarflexed from 70 to 110 at angular velocity of 1,200
• Lengthening contraction performed at a rate of 5 contractions/min for 30 mins (150R)
• Large injuries
• Ankle plantarflexed from 90 to 180 angular velocity of 1,200
• 3 minute rest between contractions (15R)
Quantifying injury and recovery
• Injury • Recovery
• Measurements were taken: • Measurements were taken:
• 10 minutes, 3 days, 7 days, 14 days, 21 • 10 minutes, 3 days, 7 days, 14 days, 21
days post injury days post injury
• Membrane damage • Maximum tetanic torque
• Uptake of FDx • Post injury compared to pre-injury
• Imaging and counting of myofibers • Membrane resealing
• Myofiber necrosis • Imaging and counting of myofibers
• Necrosis: pale and fragmented • Compared to pre-injury
myoplasm • Myogenesis
• Inflammation • Counting of CNFs and dMHC+ fibers
• Counting the number of mononuclear • Expression of developmental isoforms of
cells myosin heavy chain at 5 days post injury
Statistical Analyses
• Multifactorial ANOVA (MANOVA)
• Independent variables:
• Mouse strain
• Type of Injury
• Irradiation status
• Day of measurement
• Raw torque was analyzed with MANOVA and Tukey’s post hoc tests
• Repeated measures on time (repetitions)
• Alpha value: 0.05
Efficacy of Irradiation
• Irradiation drastically reduces CNF count induced by cardiotoxin (CTX)
• CTX induces myogenesis
• Results:
• Unirridiated muscles showed 44 (13) % dMHC+ fibers and 40 (6)% CNFs
• Irradiated muscles showed 4 (2) % dMHC+ fibers and 4 (2)% CNFs
• Contractile function is unaffected by irradiation
Large Strain Injuries (15R)
• Membrane damage
• Not affected by irradiation
• Both groups maintained the same amount of the membrane at D0 and D3
• Similar levels of FDx
• Myofiber necrosis
• Assessed at D0 and D3
• No necrosis at D0
• Significant increase in necrosis from D0 to D3 for Dysferlin-null group (p<0.00001)
• Inflammatory response
• Assessed at D0 and D3
• Dysferlin-null group expressed a significant increase of mononuclear cells at D3
• Irradiation did not induce inflammation
• Contributed by limiting myogenesis
 Recovery from large strain injuries
• All groups: torque reduction at D0
• Compared to pre-injury
• Dysferlin + No-Irradiation:
• Back to pre-injury levels at D3 (p<0.00001)
• Dysferlin + Irradiation:
• Back to pre-injury levels at D3 (p<0.00001)
• Dysferlin-null + No-Irradiation:
• Back to pre-injury levels at D14
• Dysferlin-null + Irradiation:
• No recovery in 21 days
 Recovery from large strain injuries
• Membrane re-sealing
• Dysferlin-null group significantly loses membrane by D7
• Dysferlin group maintains membrane
 Recovery from large strain injuries
• Myogenesis
• Peaked at D14
• Does not occur in the presence of dysferlin
• Occurs in the dysferlin-null group
• Increase in CNF
• Reduced expression of dMHC
Small Strain Injuries (150R)
• Membrane damage
• Not affected by irradiation
• More membrane maintained from D0 to D3 compared to 15R for both groups
• Myofiber necrosis
• Low necrosis at D0
• Both groups showed increases in necrosis at D3
• Significant increase in necrosis for dysferlin-null group at D3 (p<0.0001) compared to
dysferlin group
• Inflammatory response
• Assessed at D0 and D3
• Both groups expressed a significant increase of mononuclear cells at D3
• Irradiation did not induce inflammation
• Contributed by limiting myogenesis
Recovery from small strain injuries
• All groups: torque reduction at D0
• Compared to pre-injury
• Dysferlin + No-Irradiation:
• Significant improvement from D7 to D14
• Back to pre-injury levels at D21
• Dysferlin + Irradiation:
• No recovery in 28 days
• Dysferlin-null + No-Irradiation:
• Back to pre-injury levels at D28
• Dysferlin-null + Irradiation:
• No recovery in 28 days
• Was worse than Dysferlin + Irradiation
Recovery from small strain injuries
• Membrane re-sealing
• Significant loss of the membrane from D3 to D7 for both groups
Recovery from small strain injuries
• Myogenesis
• Peaked at D14
• Occurs in both groups
• Increase in CNF
• Reduced expression of dMHC
• Significantly more occurrence in 15R analogs
Effects of dysferlin on recovery
• Dysferlin does not affect the severity of injuries
• Dysferlin is needed for the rapid recovery of small and large injuries
• Lack of dysferlin does not inhibit myogenesis
• Dysferlin possibly plays an upstream anti-inflammatory role in
recovery
• Study by Millay et. al showed that expression of dysferlin is able to reduce the
degenerative characteristics of dysferlinopathies
Effects of myogenesis on recovery
• Recovery from large injury does not involve myogenesis
• Recovery from multiple small injuries requires myogenesis
Discussion
• Limitations:
• The health of the recovered muscle was unassessed
• Structural integrity of the membrane
• Implications of research:
• Therapies targeted at the secondary response of muscle injury
• Target macrophage infiltration
• Future studies:
• Determine the exact role which dysferlin plays in rapid muscle recovery
• Study the way dysferlin retains and excludes molecules
References
• Vernengo L, Carrasco L, Angelini C, Rodriguez MM (2013)
Dysferlinopathies. J Genet Syndr Gene Ther 4:134. doi: 10.4172/2157-
7412.1000134
• Lovering RM, Roche JA, Bloch RJ, De Deyne PG (2007) Recovery of
function in skeletal muscle following 2 different contraction-induced
injuries. Arch Phys Med Rehabil 88: 617–625