MMex38 mice harbor the murine dysferlin mutation DYSFL1360P synonymous to the dysferlinopathy patient missense mutation DYSFL1341P. We previously found that 4-PBA could increase plasma membrane localization and restore membrane repair function to of DYSFL1341P in vitro and could also do the same after a 2-day treatment of MMex38 mice in vivo (Tominaga et. al. 2021, iScience). During this grant period we began a study to determine if the clinically approved drug, sodium phenylbutyrate (4-PBA), is efficacious in preventing or reducing the severity of dystrophic phenotypes in the MMex38 mouse model of dysferlinopathy.
We first undertook an aging study of MMex38 mice in order to establish a baseline for histological and physiological changes that occur over time in this model in order to be able to establish endpoints for a longitudinal study of 4-PBA. To these ends, we established an aging colony of MMex38 (MUT) and wildtype (WT) control animals ranging from 3 months of age to up to 2 years of age, and isolated hind limb muscles from at least three male mice from each age group; including Quadriceps (Q), Gluteus (GL), Gastrocnemius (GC), Tibialis Anterior (TA), Psoas (P), and Extensor Digitorum Longus (EDL) muscles. We found statistically significant reductions in the weights of P, GL, and Q muscles from MUT animals at or over 12 months, and in GC muscles in MUT mice at or over 16 months. TA muscle show weight reduction at or over 20 months of age, while no differences were noted in EDL. We prepared fixed and fresh frozen tissue samples from all muscle groups for histological analysis, including H&E staining, Mason’s Trichrome staining, Congo Red staining, and Dysferlin immunohistochemistry in order to quantify dystrophic histological markers common to dysferlinopathy such as reduced myofiber size, increased numbers of central nuclei caused by regenerating myofibers, increased fat and collagen content/distribution indicative of fat infiltration and fiber replacement, and fibrosis. Most of these samples have been processed and analysis underway to determine differences between the various WT and MUT muscle groups.
To access the physiological effects on hind limb muscles, we developed an in-house Inclined Balance Beam (IBB) assay that scores the amount of time it takes a mouse to traverse a specified distance on inclined beams of various widths (24mm, 12mm, 10mm, 8mm). We found statistically significant differences in cross times between WT and MUT animals at 13 and 14 months of age on 12mm, 10mm, and 8mm width beams. We therefore plan to use the balance beam assay as a measure of muscle function in our longitudinal 4-PBA drug study.
During this granting period we established a longitudinal 4-PBA drug treatment trial. Thirty-two 2-month-old individually caged MMEx38 animals (16 male and female), were placed on a drug treatment protocol where half receive water containing 4-PBA (2mg/mL) and the remaining are given untreated water ad libitum, with water changed weekly. Animals were placed on protocol as they aged in; full enrollment occurred over three months. All animals were found to consume equivalent amounts of water, and showed equivalent increases in body weight over time; no adverse health events were found. Animals have been/are trained for IBB at 10 months of age, and assayed on the IBB each month thereafter. During our next grant period, we plan to end the longitudinal 4-PBA drug study after IBB assay are performed at 14 months of age, whereupon the various muscle groups listed above will be isolated, weighed, and processed for muscle histology.