STUDY OF SKELETAL MUSCLE FUNCTION PATHOMECHANISMS IN DUCHENNE MUSCULAR DYSTROPHY (DMD) MOUSE MODEL AFTER DYSTROPHIN RESTORATION BY CELL TRANSPLANTATION

C.K. Bourgeois Yoshioka^1,2, N. Takenaka-Ninagawa¹, M. Goto¹, M. Miki^1,2, T. Aoyama², H. Sakurai¹

¹Center for iPS Cell Research and Application, Kyoto University, Kyoto, Japan, ²Kyoto University, Graduate School of Medicine, Human Health Sciences, Department of Physical Therapy, Kyoto, Japan

Background: The world most common form of muscular dystrophies, Duchenne muscular dystrophy (DMD), is caused by several mutations to the X-linked DMD gene, known to code for dystrophin protein. Loss of dystrophin and progressive degeneration of skeletal muscle fibers have been reported as molecular phenotypes while muscle weakness and muscle fatigue are responsible for progressive degradation of the gait and cardiorespiratory disorders from early childhood. However, the link between loss of dystrophin protein, muscle molecular dysfunctions and DMD functional phenotype remains unknown.

Purpose: This study aims to clarify DMD mice skeletal muscle function biomolecular pathomechanisms using cell transplantation to supplement dystrophin protein into DMD muscles.

Methods: Gastrocnemius intramuscular transplantations of dystrophin expressing immortalized human myoblast cells (Hu5/KD3) (Shiomi et al. 2011) in an immunodeficient DMD mouse model (DMD-null/NSG) (Kudoh et al. 2005, Zhao et al. 2020) were conducted to create dystrophin supplemented-DMD mice muscles. Electrical stimulations and treadmill were used to evaluate DMD mouse muscle strength (plantar flexion isometric contraction torque (Itoh et al. 2017)) and muscle fatigue (plantar flexion maximal contraction torques ratio before and after muscle fatigue created by treadmill running), while immunohistochemistry and western blotting methods were performed to analyze several key proteins expressions.

Results: Muscle fatigue ratio evaluation showed significant improvement in dystrophin supplemented-DMD mice muscles compared to DMD control, although plantar flexion maximal contraction torque was found statistically similar in both groups. The number of dystrophin positive myofibers in transplanted DMD muscles was statistically correlated to the plantar flexion maximal contraction torque, but not to muscle fatigue ratio. Histological analysis of DMD transplanted muscles revealed that damaged myofibers, positive to Evans blue dye (EBD), were significantly reduced compared to DMD control, and none of dystrophin positive myofibers in DMD transplanted muscles was EBD positive. Furthermore, myosin heavy chains type I and IIa were highly expressed in transplanted DMD muscles, compared to DMD control. As preliminary studies have shown that mitochondrial activity and ATP production in DMD mice muscles are likely to be related to DMD muscle molecular dysfunctions as well, detailed validation experiments are currently under way.

Conclusions: Dystrophin supplementation by cell transplantation in DMD mice skeletal muscles enhanced their tolerance to muscle fatigue. Results indicated that dystrophin protein could have a protective role against muscle damage created by forced running. However, the number of supplemented dystrophin positive fibers alone could not explain that functional improvement. Further analysis demonstrated that one explanation was that newly regenerated myofibers from transplanted cells were slow-twitch type fibers. Study of metabolic impairment in those transplanted DMD mice muscles is still ongoing to further clarify DMD muscle function pathomechanisms.

Implications: Regenerative rehabilitation is an emerging field applying regenerative medicine advanced technologies to rehabilitation sciences. This research will not only clarify the still poorly understood DMD muscle pathogenesis in mouse model but also the physiological effects of cell transplantation therapeutical strategy towards clinical application. In the long term, a better understanding of cellular processes and mechanical loading effects on DMD mice muscles will also result in a more informed, effective and safe rehabilitation management of DMD patients.

Funding acknowledgements: N/A

Keywords:
Duchenne muscular dystrophy
Cell transplantation
Muscle function evaluation

Topics:
Paediatrics
Musculoskeletal

Did this work require ethics approval? Yes

Institution: Kyoto University

Committee: Kyoto University Animal Experiment Committee

Ethics number: 17-81-7