Endurance exercise leads to beneficial molecular and physiological effects in a mouse model of myotonic dystrophy type 1

Abstract

Introduction: Myotonic dystrophy type 1 (DM1) is a multisystemic disease caused by expansion of a CTG repeat in the 3' UTR of the Dystrophia Myotonica-Protein Kinase (DMPK) gene. While multiple organs are affected, more than half of mortality is due to muscle wasting.

Methods: It is unclear whether endurance exercise provides beneficial effects in DM1. Here, we show that a 10-week treadmill endurance exercise program leads to beneficial effects in the HSA^LR mouse model of DM1.

Results: Animals that performed treadmill training displayed reduced CUGexp RNA levels, improved splicing abnormalities, an increase in skeletal muscle weight and improved endurance capacity.

Discussion: These results indicate that endurance exercise does not have adverse effects in HSA^LR animals and contributes to beneficial molecular and physiological outcomes.

Keywords: endurance exercise; mouse model; myotonic dystrophy type I; skeletal muscle; splicing; treadmill.

FIGURE 1

HSA^LR mice were subjected to endurance exercise training. A, The transgene for HSA^LR mice contains a genomic segment of the HSA gene with an expanded CTG trinucleotide repeat (250 repeats) inserted into the 3’- UTR of the last exon. B, Adult HSA^LR (FvB) or age-matched WT FvB mice were randomly assigned to sedentary or exercised groups. The 10-week exercise protocol is outlined. Muscle was harvested at the conclusion of week 10 for muscle weights, histology, and molecular analysis (repeat RNA levels, splicing, MBNL levels)

FIGURE 2

HSA^LR exercised animals display a significant decrease in CUGexp RNA levels. Total RNA was extracted from gastrocnemius muscle of adult male animals, either homozygous for the HSA^LR transgene or age-matched male nontransgenic WT controls. Animals in the exercise group were maintained on a 10-week treadmill exercise training protocol 5 days per week as indicated in Figure 1B. All RNA was extracted at the conclusion of the 10-week study. RT-PCR was performed using primers in exons 5 and 6 of hACTA1 (upstream of repeats) (A) or exons 6/7 and exon 8 of mActa1 (B), and normalized to Gapdh. The primer pairs are specific for the human or mouse ACTA1/Acta1 gene. n = 5 per group (n = 6 for HSA^LR exercised). Statistical analysis was conducted by one-way ANOVA. Post-hoc analysis was performed using Tukey’s HSD correction. Error bars indicate ± SD. ****P < 0.0001

FIGURE 3

HSA^LR exercised animals display significant changes in alternative splicing. Total RNA was extracted from gastrocnemius muscle at the conclusion of the 10-week study. Standard RT-PCR was performed using primers to the constitutive exons flanking the alternative exons. Ratios of exon inclusion were quantified as: [intensity of top exon inclusion band/ (intensity of the top + bottom exon skipping band]) × 100, displayed as PSI. A, Representative RT-PCR of Atp2a1 exon 22 alternative splicing event illustrating decreased exon inclusion in HSA^LR sedentary animals in comparison to FVB controls. A dramatic transition in the splicing pattern of Atp2a1 was observed in exercised HSA^LR animals toward the WT adult splicing pattern as compared to HSA^LR sedentary animals. B, Quantification of alternative splicing events showing a transition in several splicing events in HSA^LR exercised animals (n = 5 per group, n = 6 for HSA^LR exercised). Statistical analysis was conducted with two-way ANOVA. Post-hoc analysis was performed using Tukey’s HSD correction. Error bars indicate ± SD. *P < .05, **P < .01, ****P < .0001

FIGURE 4

HSA^LR exercised animals display an increase in muscle weight with no changes in body weight. Adult WT male FvB or HSA^LR were subjected to either no exercise or treadmill training for a period of 10 weeks. A, Body weight was measured throughout the time course. B, Muscle weights were measured at the conclusion of week 10 and normalized to tibia length. Mean for the group ± SD is displayed for each time point. Statistical analysis was conducted with two-way ANOVA. Post-hoc analysis was performed using Tukey’s HSD correction. Error bars indicate ± SD. *P < .05, **P < .01, ***P < .001

FIGURE 5

Exercised animals display significant decreases in body fat in HSA^LR and WT animals. Body fat percentage (A) and bone mineral density (B) was measured at the start of the study (start of week 1), midway through (end of week 5), and at the conclusion of the training protocol (end of week 10). Individual data points displayed with mean ± SD. Statistical analysis was conducted with two-way ANOVA. Post-hoc analysis was performed using Tukey’s HSD correction. *P < .05, **P < .01, ****p < .0001

FIGURE 6

Exercised animals from HSA^LR or WT display significant changes in the distance run on exhaustive treadmill with no significant changes in grip strength. A, Maximum distance run on exhaustive treadmill was measured at the start of week 1 and the conclusion of week 10 with exercised animals displaying an increased time until exhaustion in both groups. B, VO_2max was measured with exercised animals from both groups displaying a comparable increase in VO_2max. C, Grip strength of front limbs measured at the conclusion of week 10 normalized to body weight shows no statistically significant changes in force although there is variability among HSA^LR exercised animals. We did observe two HSA^LR animals with substantially reduced grip strength. Statistical analysis was conducted by two-way ANOVA followed by Tukey’s HSD correction (A) or Kruskal-Wallis test (C). Error bars indicate ±SD. ****P < .0001

FIGURE 7

Exercised animals display a decrease in Mbnl2 protein levels. Total protein was isolated from gastrocnemius muscle at the conclusion of the 10-week study. Protein levels of Mbnl1 and Mbnl2 were detected by Western blot and normalized to total protein as determined by Coomassie (representative Coomassie shown). Statistical analysis was conducted using an unpaired t-test. Error bars indicate ± SD. *P < .05