Characterizing the Role of Phospholamban in Duchenne Muscular Dystrophy
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Duchenne muscular dystrophy (DMD) and the murine model, mdx, are recessive X-linked myopathies characterized by aberrant Ca2+-handling resulting in muscle atrophy and weakness. Phospholamban (PLN) is a protein inhibitor of sarco(endo)plasmic reticulum Ca2+-ATPases (SERCAs) that physically interacts with SERCA to regulate Ca2+-handling. Targeted therapy to improve SERCA function is a proven strategy to alleviate DMD in mdx mice. In this study, Pln-/- mice were crossed with mdx mice to generate mdx/Pln-/- double knockout mutant mice. Since PLN inhibits SERCAs, it was hypothesized that PLN ablation would mitigate Ca2+ dysregulation and rescue the dystrophic phenotype. Soleus and diaphragm muscles from WT, mdx/Pln+/+ and mdx/Pln-/- mice were excised to determine differences in the muscle morphology and functionality. Histological analysis revealed stark increases in the proportion of centralized nuclei and collagen invasion in mdx/Pln-/- and mdx mice compared to WT, however, there were no differences in these markers between mdx groups. Immunofluorescence staining demonstrated that both soleus and diaphragm from mdx/Pln-/- mice shifted towards type IIB and type IIX fibre types, as the proportion of these fibres were significantly greater than mdx/Pln+/+ and WT. This shift was accompanied by increased cross sectional area of type IIB fibres in mdx/Pln-/- compared to mdx/Pln+/+. Western blotting analysis of soleus and diaphragm muscle homogenate showed an increase inexpression of the SERCA regulator, sarcolipin (SLN), in both mdx groups relative to WT, however, there was a significant decline in SLN content in mdx/Pln-/- compared to mdx/Pln+/+. Additionally, there was a significant elevation in PLN content in mdx/Pln+/+ relative to WT in the soleus muscle. The solei of mdx/Pln+/+ and mdx/Pln-/- mice exhibited a significant reduction in force production compared to their WT counterparts at all frequencies when normalized to cross sectional area. Unexpectedly, the force generated by mdx/Pln-/- soleus was significantly decreased at all stimulation frequencies when compared to mdx/Pln+/+. Furthermore, there was a significant reduction in soleus and diaphragm Ca2+ uptake in both the mdx/Pln+/+ and mdx/Pln-/- groups compared to WT, and surprisingly the rate of Ca2+ uptake was significantly lower in mdx/Pln-/- muscles compared with mdx/Pln+/+. Interestingly, while there were differences in SERCA mediated Ca2+ uptake between experimental groups, there were no significant differences in Ca2+- ATPase activity between WT, mdx/Pln+/+ and mdx/Pln-/- groups in the soleus and diaphragm muscles. Overall, these results demonstrate that PLN ablation in the mdx mouse model resulted in a worsening of the disease phenotype, as evident by elevations in centralized nucleation, a reduction in the ability to generate force and impairments in SERCA mediated Ca2+ uptake. These results suggest that PLN could potentially provide stabilization of the SERCA structure and function during oxidative stress.
Cite this version of the work
Emma Sara Juracic (2018). Characterizing the Role of Phospholamban in Duchenne Muscular Dystrophy. UWSpace. http://hdl.handle.net/10012/12868