Generation of desminopathy in rats using CRISPR‐Cas9

Abstract

Background Desminopathy is a clinically heterogeneous muscle disease caused by over 60 different mutations in desmin. The most common mutation with a clinical phenotype in humans is an exchange of arginine to proline at position 350 of desmin leading to p.R350P. We created the first CRISPR-Cas9 engineered rat model for a muscle disease by mirroring the R350P mutation in humans. Methods Using CRISPR-Cas9 technology, Des c.1045-1046 (AGG > CCG) was introduced into exon 6 of the rat genome causing p.R349P. The genotype of each animal was confirmed via quantitative PCR. Six male rats with a mutation in desmin (n = 6) between the age of 120-150 days and an equal number of wild type littermates (n = 6) were used for experiments. Maximal plantar flexion force was measured in vivo and combined with the collection of muscle weights, immunoblotting, and histological analysis. In addition to the baseline phenotyping, we performed a synergist ablation study in the same animals. Results We found a difference in the number of central nuclei between desmin mutants (1 ± 0.4%) and wild type littermates (0.2 ± 0.1%; P < 0.05). While muscle weights did not differ, we found the levels of many structural proteins to be altered in mutant animals. Dystrophin and syntrophin were increased 54% and 45% in desmin mutants, respectively (P < 0.05). Dysferlin and Annexin A2, proteins associated with membrane repair, were increased twofold and 32%, respectively, in mutants (P < 0.05). Synergist ablation caused similar increases in muscle weight between mutant and wild type animals, but changes in fibre diameter revealed that fibre hypertrophy in desmin mutants was hampered compared with wild type animals (P < 0.05). Conclusions We created a novel animal model for desminopathy that will be a useful tool in furthering our understanding of the disease. While mutant animals at an age corresponding to a preclinical age in humans show no macroscopic differences, microscopic and molecular changes are already present. Future studies should aim to further decipher those biological changes that precede the clinical progression of disease and test therapeutic approaches to delay disease progression.

Increased remodeling and impaired adaption to endurance exercise in desminopathy

“…Given this stark decrease in muscle function, it was somewhat surprising to see only moderate histopathological changes after running in the DES animals. The R349P animals demonstrated a higher percentage of fibers with central nuclei in DES compared to WT rats in the sedentary condition, which is in line with our previous findings in this strain 26 . However, this percentage did not go up in DES animals after the downhill running protocol, but rather tended to be reduced.…”

“…More recently, we engineered a CRISPR-Cas9 knock-in rat with the same mutation to create a model organism that is physiologically closer to humans. While we saw a clear histopathological phenotype with sarcoplasmic aggregates and signs of regeneration in the muscle of 6-month-old mutants, muscle mass and force did not differ significantly from wildtype littermates even after synergist ablation 26 . The observed histopathological and molecular indicators of injury and regeneration were exacerbated by an acute eccentric contraction challenge in older animals 27 .…”

“…In previous studies we investigated histopathological and molecular changes in our model at a preclinical age after functional overload and in old rats after acute eccentric loading. We showed that mutant animals at an age corresponding to a preclinical age in humans had a mild myopathy and increased markers for muscle injury and repair 26 . To see whether ageing exacerbates the phenotype, in our next study we looked at 400-500-day old animals in conjunction with exercise.…”

Annexins and Membrane Repair Dysfunctions in Muscular Dystrophies

“…Overexpression of ANXA2 is also observed in DMD, Becker muscular dystrophy or LMGDR12 and shedding of ANX-positive vesicles have been shown in ANO5-knockout myofibers (LMGDR12), suggesting these diseases may result from fibrotic or adipogenic replacement of myofibers [ 54 , 129 ]. Recently, an increase of 32% in the expression of ANXA2 has been also observed in a rat model of desminopathy [ 133 ].…”

Nuclear Mechanotransduction in Skeletal Muscle

“…Accumulating evidence indicates that desmin is also crucial as a stress-transmitting and stress-signaling network [98,[107][108][109][110]. Desmin interactions with the nucleus are required to maintain nuclear architecture in cardiomyocytes [111] and to prevent nuclear and muscle damage in response to mechanical challenges [111,112]. Future studies will determine the contribution of desmin scaffolds in myonucleus architecture and function.…”