Current knock out models of Calpain3 deficiency on a C57BL/6J genetic background are relatively mild in their phenotype with only minor histological deficits. We created and characterized Calpain3 knockouts on four different genetic backgrounds: 129/Sv (reference strain), DBA2/J, FVB/NJ and an inbred strain from the Collaborative Cross, CC041/UncJ, with the goal to develop a LGMD2A model with a more clinically-relevant phenotype, available from a public mouse Repository.
Calpain 3 mutations were induced by CRISPR/Cas9-mediated deletions of exons 2 and 3 and verified by Western blotting. The severity of the phenotypes was compared between strains with standard assays including grip strength, rotarod, and open field testing. To elicit exercise-induced phenotypes, we also tested the mutants on the wheel running test, the forced-exercise treadmill test, and by in vivo muscle force recording with repeated eccentric muscle contractions. Serum creatine kinase was measured in all strains. Muscle histology consisted in the quantification of fibrosis, and central nucleation.
On mice aged to more than one year, we found no significant phenotype on the grip strength, rotarod, wheel-running, treadmill and open field tests. Calpain 3 knock outs in the CC041 background, although they were able to develop the same amount of force under tetanic stimulation of their muscles, presented a specific decrease in isometric muscle force upon repeated eccentric contraction. CC041-Capn3 and DBA-Capn3 mutants presented the highest elevation in serum CK, followed by FVB-Capn3 mutants. Muscle histopathology, characteristic of LGMD2A consisted in central nucleation, interstitial fibrosis and fat infiltration.
In conclusion, the severity of the Calpain 3 deficiency phenotype is sensitive to the genetic background in mice. Muscle histopathology in absence of calpain, marked by degeneration and inflammation, does not always correlate with functional deficit and exercise intolerance. This work provides the research community with new models and phenotypic tests able to support drug discovery for LGMD2A.