В6.А-Dysfprmd/GeneJ mice as a genetic model of dysferlinopathy

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Abstract

The aim of the work was behavioral and pathomorphological phenotyping of the mice knockout for the DYSF gene, which plays an important role in the development and progression of dysferlinopathy.

Materials and methods. A B6.A-Dysfprmd/GeneJ (Bla/J) mice subline was used in the work. During the study, a muscle activity was determined basing on the following tests: “Inverted grid”, “Grip strength”, “Wire Hanging”, “Weight-loaded swimming”, Vertical Pole”. Histological and immunofluorescent examinations of skeletal muscles (m. gastrocnemius, m. tibialis) were performed. The presence and distribution of the dysferlin protein was assessed, and general histological changes in the skeletal muscle characteristics of mice at the age of 12 and 24 weeks, were described. A morphometric analysis with the determination of the following parameters was performed: the proportion of necrotic muscle fibers; the proportion of fibers with centrally located nuclei; the mean muscle fiber diameter.

Results. The “Grip strength” test and the “Weight-loaded swimming” test revealed a decrease in the strength of the forelimbs and endurance in the studied mice of the Bla/J subline compared to the control line. The safety of physical performance was checked using the “Wire Hanging” test and the “Vertical Pole” test, which showed a statistically significant difference between the studied mice and control. The coordination of movements and muscle strength of the limbs examined in the “Inverted Grid” test did not change in these age marks. Decreased grip strength of the forelimbs, decreased physical endurance with age, reflects the progression of the underlying muscular disease. Histological methods in the skeletal muscles revealed signs of a myopathic damage pattern: necrotic muscle fibers, moderate lympho-macrophage infiltration, an increase in the proportion of fibers with centrally located nuclei, and an increase in the average fiber diameter compared to the control. The dysferlin protein was not found out in the muscle tissues.

Conclusion. Taking into account the results of the tests performed, it was shown that the absence of Dysf-/- gene expression in Bla/J subline mice led to muscular dystrophy with the onset of the development of phenotypic disease manifestations at the age of 12 weeks and their peak at 24 weeks. Histopathological phenotypic manifestations of the disease are generally nonspecific and corresponded to the data of intravital pathoanatomical examination in diferlinopathy patients. The mice of the studied subline Bla/J are a representative model of dysferlinopathy and can be used to evaluate new therapeutic agents for the treatment of this disease.

About the authors

Mikhail V. Korokin

Belgorod State National Research University

Author for correspondence.
Email: korokin@bsu.edu.ru
ORCID iD: 0000-0001-5402-0697

Doctor of Sciences (Medicine), Associate Professor of the Department of Pharmacology and Clinical Pharmacology

Russian Federation, 85, Pobedy Str., Belgorod, 308015

Elena V. Kuzubova

Belgorod State National Research University

Email: 1015artek1015@mail.ru
ORCID iD: 0000-0003-2425-5027

Junior Researcher, Laboratory of Genetic Technologies and Gene Editing for Biomedicine and Veterinary Medicine

Russian Federation, 85, Pobedy Str., Belgorod, 308015

Alexandra I. Radchenko

Belgorod State National Research University

Email: sandrinkaradchenko@gmail.com
ORCID iD: 0000-0002-4554-2116

Junior Researcher of Laboratory of Genetic Technologies and Gene Editing for Biomedicine and Veterinary Medicine

Russian Federation, 85, Pobedy Str., Belgorod, 308015

Roman V. Deev

North-Western State Medical University named after I.I. Mechnikov; PJSC “Human Stem Cells Institute”

Email: romdey@gmail.com
ORCID iD: 0000-0001-8389-3841

Candidate of Sciences (Medicine), Associate Professor, Acting Head of the Department of Pathological Anatomy, Director for Science

Russian Federation, 41, Kirochnaya Str., St. Petersburg, 191015; Bld. A, 10, 60-Letiya Oktyabrya Ave., Moscow, 117292

Ivan A. Yakovlev

LLC “Genotarget”

Email: ivan@ivan-ya.ru
ORCID iD: 0000-0001-8127-4078

General Director

Russian Federation, 48/7, Nobel Str., Skolkovo Innovation Center, Moscow, 121205

Aleksey V. Deikin

Belgorod State National Research University

Email: alexei@deikin.ru
ORCID iD: 0000-0001-9960-0863

Candidate of Sciences (Biology), Associate Professor of the Department of Pharmacology and Clinical Pharmacology

Russian Federation, 85, Pobedy Str., Belgorod, 308015

Nikita S. Zhunusov

Belgorod State National Research University

Email: nzhunu@mail.ru
ORCID iD: 0000-0002-1969-3615

Assistant of the Department of Pharmacology and Clinical Pharmacology

Russian Federation, 85, Pobedy Str., Belgorod, 308015

Anastasia M. Krayushkina

Belgorod State National Research University

Email: krayushkina_2016@mail.ru
ORCID iD: 0000-0002-6830-3820

Assistant of the Department of Pharmacology and Clinical Pharmacology

Russian Federation, 85, Pobedy Str., Belgorod, 308015

Vladimir M. Pokrovsky

Belgorod State National Research University

Email: pokrovskiy@bsu.edu.ru
ORCID iD: 0000-0002-1493-3376

Assistant of the Department of Pharmacology and Clinical Pharmacology

Russian Federation, 85, Pobedy Str., Belgorod, 308015

Olesya A. Puchenkova

Belgorod State National Research University

Email: lesya759@yandex.ru
ORCID iD: 0000-0002-7657-0937

Resident of the Medical Institute

Russian Federation, 85, Pobedy Str., Belgorod, 308015

Kirill D. Chaprov

Belgorod State National Research University; Institute of Physiologically Active Compounds at Federal Research Center of Problems of Chemical Physics and Medicinal Chemistry, Russian Academy of Sciences (IPAC RAS)

Email: chapkir@gmail.com
ORCID iD: 0000-0002-0258-1879

Junior Researcher of Laboratory for Modeling and Gene Therapy of Human Diseases, Junior Researcher

Russian Federation, 85, Pobedy Str., Belgorod, 308015; 1, Severny Driveway, Chernogolovka, 142432

Natalya V. Ekimova

Belgorod State National Research University

Email: ekimova@bsu.edu.ru
ORCID iD: 0000-0002-3629-048X

Assistant of the Department of Pharmacology and Clinical Pharmacology

Russian Federation, 85, Pobedy Str., Belgorod, 308015

Sergey N. Bardakov

S. M. Kirov Military Medical Academy

Email: epistaxis@mail.ru
ORCID iD: 0000-0002-3804-6245

Neurologist, Lecturer of the Department of Nervous Diseases

Russian Federation, 6, Academician Lebedev Str., St. Petersburg, 194044

Olga N. Chernova

North-Western State Medical University named after I.I. Mechnikov

Email: olgachernova92@yandex.ru
ORCID iD: 0000-0001-6310-9319

Assistant of the Department of Human Morphology

Russian Federation, 41, Kirochnaya Str., St. Petersburg, 191015

Aleksey M. Emelin

Belgorod State National Research University; North-Western State Medical University named after I.I. Mechnikov

Email: eamar40rn@gmail.com
ORCID iD: 0000-0003-4109-0105

Assistant of the Department of Pathological Anatomy, Postgraduate Student of the Department of Pathological Anatomy

Russian Federation, 85, Pobedy Str., Belgorod, 308015; 41, Kirochnaya Str., St. Petersburg, 191015

Igor S. Limaev

North-Western State Medical University named after I.I. Mechnikov

Email: ig.limaev@gmail.com
ORCID iD: 0000-0002-0994-9787

Senior Assistant of the Department of Pathological Anatomy

Russian Federation, 41, Kirochnaya Str., St. Petersburg, 191015

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Supplementary files

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2. Figure 2 – Detection of retrotransposon presence in Bla/J mice by PCR. Note: product size WT (wild type) is 204 bps; product size Bla/J 237 – bps; NTC – negative control without template.

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3. Figure 3 – Physiological parameters of Bla/J mice. Note: the analysis of forelimbs grip strength in the “Grip strength test” (A and B), endurance in the “Weight-loaded swimming test” (C and D), physical performance in the “Wire Hanging test” (E and F) and the “Vertical Pole test” (G and H), coordination of movements and limbs muscle strength in the “Inverted Grid test” (K and L). Experimental Bla/J and control mice without a genome modification (WT) were tested at the age of 12 (A, C, E, G, K) and 24 weeks (B, D, F, H, L). Medians and a standard error of the mean are presented, the number of animals is indicated at the bottom of the corresponding column. Samples were tested for normality, and statistical significance was assessed using the Mann-Whitney U-test (**p <0.01; ***p <0.0004, ****p <0.0001).

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4. Figure 4 – Immunofluorescent reaction with antibodies to dysferlin. Note: A – C57Bl mouse; B –line Bla/J mouse. Red color – detectable dysferlin of sarcoplasmic localization, blue color – nuclei. Finishing dyeing: DAPI. Magnification ×200

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5. Figure 1 – Scheme of primers position for amplification of genomic area (wild type) and 3’LTR area of retrotransposon (mutant). Note: WT – wild type; DYSF-F – common forward primer; DYSF-R – common reverse primer; ETn-OR – forward primer homologous to 3’LTR area of retrotransposon; 5’LTR, 5’ – long terminal repeats; 3’LTR – 3’ – long terminal repeats.

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6. Figure 5 – Striated skeletal muscle tissue of studied animals. Note: A – control, C57Bl; B–D – Bla / J. 1 – endomysial and perimysial edema; 2 – fibers with centrally located nuclei; 3 – lympho-macrophage infiltration around necrotic muscle fibers; 4 – necrotic muscle fibers, incl. macrophage invasion; 5 – rounded muscle fibers. Colour: eosin. Magnification: A–C ×200, D ×400

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7. Figure 6 – Morphometric parameters of striated skeletal muscle tissue of Bla/J mice at 12 and 24 weeks of age. Note: A – proportion of necrotic muscle fibers; B – Average cross-sectional area of muscle fibers; C – proportion of central nuclear muscle fibers (CNMFs); D – proportion of connective tissue in skeletal muscle (*p < 0.05)

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Copyright (c) 2023 Korokin M.V., Kuzubova E.V., Radchenko A.I., Deev R.V., Yakovlev I.A., Deikin A.V., Zhunusov N.S., Krayushkina A.M., Pokrovsky V.M., Puchenkova O.A., Chaprov K.D., Ekimova N.V., Bardakov S.N., Chernova O.N., Emelin A.M., Limaev I.S.

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