Fibre type dependence of dysferlin protein in rodent skeletal muscle, and the distribution of dysferlin in the t-tubular or sarcolemma in rat skeletal muscle - an extension to human skeletal muscle

Fibre type dependence of dysferlin protein in rodent skeletal muscle, and the distribution of dysferlin in the t-tubular or sarcolemma in rat skeletal muscle – an extension to human skeletal muscle

Grant Duration
09/24 – 12/25

In an extension of the research we completed examining fibre type distribution of dysferlin in rodent skeletal muscle, our second Jain project performed a series of experiments using human skeletal muscle. Individual muscle fibres were collected from frozen human vastus lateralis muscle biopsies from males and females, across a variety of ages (18-75 years old). Whilst we did not find a correlation of dysferlin with aspects of metabolism in rodents, we performed similar analyses in humans to determine if the correlation first hypothesized was observed in humans. The analyses undertaken were aimed at better identifying the most appropriate pre-clinical animal for dysferlin studies. Overall, we examined if there was a correlation between dysferlin and various markers of mitochondrial content, in Type I and IIa muscle fibres. We also performed immunohistochemistry of cross-sections of human skeletal muscle to determine if the intracellular localization of dysferlin is the same in muscle, regardless of species.

When examining the abundance of dysferlin in muscle from younger and older individuals, there was no difference based on age. Further, when sex was examined, there was no effect of sex on the abundance of dysferlin in the vastus lateralis of healthy individuals.

Whilst there was ~26% more dysferlin in Type II compared with type I muscle fibres, which was similar to that seen for the mitochondrial marker, COXIV, there was no correlation between the mitochondrial marker and dysferlin abundances. When the dysferlin abundance data were compared with that found in rodent skeletal muscle, there were species differences. In human skeletal muscle, there is more dysferlin in Type IIA compared to Type I fibres, whereas the opposite was seen in rat skeletal muscle which has more dysferlin in Type I compared to Type IIA fibres. Dysferlin abundance in mouse skeletal muscle was again different from both human and rat, with no fibre type difference.

By Robyn Murphy, PhD La Trobe University|2025-09-02T22:19:09+00:00September 10th, 2023|Past Projects|Comments Off

About the Author: Robyn Murphy, PhD La Trobe University

My overall research interest is in the area of skeletal muscle in health and disease. The research of my group focuses on various aspects of skeletal muscle biochemistry in health and disease, using exercise and disease models in humans, as well as animal models. In particular, we measure proteins in segments of individual fibres allowing issues with the heterogeneity of skeletal muscle to be overcome. We also examine movement of proteins following micro-dissection of fibres, allowing us to quantitatively assess the redistribution of proteins following various interventions.

Bla/J mice (B6.A-Dysf^prmd/GeneJ)

Availability:
The Jackson Laboratory
Stock number: 012767
Strain information: http://jaxmice.jax.org/strain/012767.html
Phone: 800-422-MICE (800-422-6423)
610 Main Street, Bar Harbor, Maine 04609 USA

Please note that this strain has been cryopreserved by the Jackson Laboratories, but the Jain Foundation maintains a private colony of these mice for public use. To obtain these mice, please contact the Jain Foundation:

Jain Foundation Inc.
9706 Fourth Ave NE, Suite 101
Seattle, Washington 98115
Phone: 425-882-1492
Fax: 425-882-1050

Email: admin@jain-foundation.org

Development:
In these mice the progressive muscular dystrophy (prmd) allele from the A/J inbred strain is crossed onto the C57BL/6 genetic background. The cross was performed in the laboratory of Dr. Isabelle Richard at Genethon and the backcross generation reached N8. In collaboration with the Jain Foundation, Dr. Richard donated the strain to The Jackson Laboratory in 2010. Upon arrival, mice were bred to C57BL/6J for at least 2 generations to establish the colony.

Mutation:
An ETn retrotransposon (5-6kb) is inserted in intron 4 of the dysferlin gene.

Symptoms:
Disease onset is observed by 2 months and is characterized by the presence of centronucleated fibers and areas of inflammation. As seen with the original background A/J, mice homozygous for the prmd allele on the C57BL/6J background display an increasing number of centronucleated fibers and impairment in the majority of muscles by 4 months of age. In order of severity, the most affected muscles are psoas, quadriceps femoris, tibialis anterior, and gastrocnemius. Mice exhibit a decreased membrane repair capacity following laser wounding experiments. In an open space assay, mice cover less distance and are less active than wild-type. Mice that are homozygous for this allele are viable, fertile and normal in size.

Comparison with other disease strains:
Disease progression is similar to A/J: slightly slower than in SJL/J, Dysf-/- (Campbell), Dysf-/- (Brown), and C57BL/10.SJL mice. As in both SJL/J and Dysf-/- (Brown) mice, proximal muscles are more severely affected than distal muscles. As in Dysf-/- (Brown) mice, abdominal muscles are also affected.

Control strain(s):
C57BL/6 (for homozygotes); littermates (for heterozygotes).

References:
Lostal W; Bartoli M; Bourg N; Roudaut C; Bentaib A; Miyake K; Guerchet N; Fougerousse F; McNeil P; Richard I. 2010. Efficient recovery of dysferlin deficiency by dual adeno-associated vector-mediated gene transfer. Hum Mol Genet 19(10):1897-907.

PMID: 20154340

Below is a quick summary of the inclusion/exclusion criteria. Please review this information carefully prior to making any decision regarding participating.

Inclusion Criteria:

Must be Non-ambulant (cannot walk 10 meters in ≤ 30 sec) and age 18 years or older
Established mutations of the dysferlin gene on both alleles
Impaired muscle function but with sufficient muscle preservation to ensure muscle transfection based on magnetic resonance image of the EDB showing sufficient muscle preservation to permit transfection
Willingness of sexually active subjects with reproductive capacity to practice reliable method of contraception (If appropriate), during the first six months after gene transfer (females) or until two negative sperm samples are obtained post gene transfer (males).

Exclusion Criteria:

Active viral infection based on clinical observations or serological evidence of HIV, or Hepatitis A, B or C infection
The presence of dysferlin mutations without weakness or loss of function
Symptoms or signs of cardiomyopathy, including:
Dyspnea on exertion, pedal edema, shortness of breath upon lying flat, or rales at the base of the lungs
Echocardiogram with ejection fraction below 40%
Diagnosis of (or ongoing treatment for) an autoimmune disease
Persistent leukopenia or leukocytosis (WBC ≤ 3.5 K/µL or ≥ 20.0 K/µL) or an absolute neutrophil count < 1.5K/µL
Concomitant illness or requirement for chronic drug treatment that in the opinion of the PI creates unnecessary risks for gene transfer
Pregnancy
AAVrh74 or AAV8 binding antibody titers > 1:50 as determined by ELISA immunoassay
Abnormal laboratory values in the clinically significant range in the table below, based upon normal values in the Nationwide Children's Hospital Laboratory: GGT, Total Bilirubin, Cystatine, Hemoglobin, White Blood Cells
This first phase of the trial will be small and will only evaluate safety; patients enrolled are not expected to show improvement in their symptoms. If the initial trial is successful, a Phase 2 trial will be scheduled, which will look for efficacy (improvement in symptoms) as well as safety. Each phase would include more patients. So, it is a process and we are pacing ourselves as we support this major milestone in the field of therapeutic intervention for people suffering from dysferlinopathy.