Sarcoplasmic Reticulum Ca ²⁺ Handling in Gastrocnemius Muscles from 10‐week‐old C57 and D2‐ <i>mdx</i> Mice

Background Duchenne Muscular Dystrophy (DMD) is an X‐linked muscle wasting disease caused by an absence of dystrophin. Secondary mechanisms including elevated myoplasmic Ca 2+ as a result of dysfunctional sarcoendoplasmic reticulum Ca 2+ ‐ATPase (SERCA) pumps and leaky ryanodine receptors (RyR) can perpetuate disease pathology. Although the widely used preclinical C57BL/10 (C57) mdx mouse model displays moderate muscle damage and weakness, this model falls short in its ability to recapitulate the more severe human phenotype. Conversely, the DBA/2J (D2) mdx mouse model displays severe and early‐onset muscle weakness, damage and histopathology; and therefore, more closely mimics human DMD. To date, there has been no characterization of muscle Ca 2+ handling in the D2‐ mdx mouse. Thus, we examined sarcoplasmic reticulum (SR) Ca 2+ handling in both D2‐ mdx and C57‐ mdx mice. Methods Eight‐week‐old male C57‐WT, C57‐ mdx , D2‐WT and D2‐ mdx mice were purchased from Jackson Laboratories. All mice underwent a hangwire test and were temporarily housed (48 hr) in a Promethion Metabolic Cage System to measure energy expenditure and cage activity. At 10 weeks of age, mice were euthanized, and gastrocnemius muscles were collected to measure Ca 2+ uptake and leak via an Indo‐1 fluorimetric assay. Serum creatine kinase (CK) activity was measured using a commercially available kit. Results Consistent with previous literature, serum CK was elevated in both C57‐ mdx and D2‐ mdx mice compared with WT, but to a greater extent in the former (8.9‐fold vs 3.3 fold, p = 0.04). While both C57‐ mdx and D2‐ mdx mice had lowered hangwire time, these impairments were more severe in the D2‐ mdx mouse (‐30% in C57‐ mdx vs ‐50% in D2‐ mdx , p = 0.09). Similarly, while both C57‐ mdx (‐15%) and D2‐ mdx (‐33%) mice had lowered total cage activity, the D2‐ mdx mice were most affected ( p = 0.03). Interestingly, metabolic cage analyses further revealed that D2‐ mdx (1.3‐fold increase) but not C57‐ mdx mice, had significantly elevated daily energy expenditure compared to their respective WT groups. When examining SR Ca 2+ uptake, we found that the D2‐ mdx mice had significantly elevated starting Ca 2+ levels compared with D2‐WT mice (5500 vs 2300 nM). When activated by ATP, there was significantly less removal of Ca 2+ in the D2‐ mdx muscles compared with WT (2.6‐fold greater area‐under‐the‐curve). However, none of these effects were observed in the C57‐ mdx mice. Furthermore, when SERCA was inhibited with cyclopiazonic acid, the rate of SR Ca 2+ leak (‐50%) and the amount of SR Ca 2+ released back into the cytosol (‐48%) were significantly lowered in the D2‐ mdx but not in the C57‐ mdx mice. This is likely reflective of impaired SR Ca 2+ uptake and filling. Conclusions Unlike the C57‐ mdx , the D2‐ mdx mice displayed early onset muscle weakness with lowered cage ambulation and hangwire time when compared with their respective WT groups. These findings were associated with impaired SR Ca 2+ handling in the D2‐ mdx mice but not the C57‐ mdx mice. Together, the extensive muscle damage and impaired SR Ca 2+ handling in the D2‐ mdx mice likely enhances their daily energy expenditure.

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