Sarcolipin deletion exacerbates soleus muscle atrophy and weakness in phospholamban overexpressing mice

<div><p>Sarcolipin (SLN) and phospholamban (PLN) are two small proteins that regulate the sarco(endo)plasmic reticulum Ca²⁺-ATPase pumps. In a recent study, we discovered that <i>Pln</i> overexpression (<i>Pln</i>^OE) in slow-twitch type I skeletal muscle fibers drastically impaired SERCA function and caused a centronuclear myopathy-like phenotype, severe muscle atrophy and weakness, and an 8 to 9-fold upregulation of SLN protein in the soleus muscles. Here, we sought to determine the physiological role of SLN upregulation, and based on its role as a SERCA inhibitor, we hypothesized that it would represent a maladaptive response that contributes to the SERCA dysfunction and the overall myopathy observed in the <i>Pln</i>^OE mice. To this end, we crossed <i>Sln</i>-null (<i>Sln</i>^KO) mice with <i>Pln</i>^OE mice to generate a <i>Pln</i>^OE/<i>Sln</i>^KO mouse colony and assessed SERCA function, CNM pathology, <i>in vitro</i> contractility, muscle mass, calcineurin signaling, daily activity and food intake, and proteolytic enzyme activity. Our results indicate that genetic deletion of <i>Sln</i> did not improve SERCA function nor rescue the CNM phenotype, but did result in exacerbated muscle atrophy and weakness, due to a failure to induce type II fiber compensatory hypertrophy and a reduction in total myofiber count. Mechanistically, our findings suggest that impaired calcineurin activation and resultant decreased expression of stabilin-2, and/or impaired autophagic signaling could be involved. Future studies should examine these possibilities. In conclusion, our study demonstrates the importance of SLN upregulation in combating muscle myopathy in the <i>Pln</i>^OE mice, and since SLN is upregulated across several myopathies, our findings may reveal SLN as a novel and universal therapeutic target.</p></div

Sarcolipin deletion exacerbates soleus muscle atrophy and weakness in phospholamban overexpressing mice - Fig 5

<p>Elevated NFATc1 phosphorylation status (A) and a failure to promote stabilin-2 expression (B) indicates blunted calcineurin signaling in <i>Pln</i>^OE/<i>Sln</i>^KO mice. * Significantly different from WT using a one-way ANOVA with a Games-Howell post-hoc for unequal variances (A, n = 7–12 per genotype), or a Tukey’s post-hoc test (B, n = 9–10 per genotype); ^†significantly different from <i>Pln</i>^OE, <i>p</i> ≤ 0.05. All values are means ± SEM.</p

SLN does not contribute to CNM pathology in <i>Pln</i>^OE mice.

<p>Representative H&E (A), immunofluorescent (B), and SDH (C) stained sections showing the CNM phenotype of elevated central nuclei (D), type I fiber predominance (E) and type I fiber hypotrophy and type II fiber hypertrophy (F), and central aggregation of oxidative activity (yellow arrows in C) (n = 4–6 per genotype). (G) Transmission electron micrographs illustrating triad disruptions with swollen sarcoplasmic reticulum in both the <i>Pln</i>^OE and <i>Pln</i>^OE/<i>Sln</i>^KO soleus muscles. Arrowheads point to the swollen sarcoplasmic reticulum with corresponding area. Van-Giesons staining (H) quantified with imageJ (I) reveals greater endomysial fibrosis the <i>Pln</i>^OE and <i>Pln</i>^OE/<i>Sln</i>^KO soleus muscles compared with WT (n = 4 per genotype). (J) Representative SDH-stained and H&E-stained sections displaying core-like aspects in the <i>Pln</i>^OE and <i>Pln</i>^OE/<i>Sln</i>^KO soleus muscles that cannot be explained by the presence of an artifact or vacuole. Arrows point to the core-like aspect and asterisks depict the same fiber within serial sections. *Significantly different from WT using a one-way ANOVA with a Tukey’s post-hoc, <i>p</i> ≤ 0.05. All values are means ± SEM. CSA, cross-sectional area. All scale bars are set to 50 μm except for (G) where scale bars are set to 100 nm.</p

<i>Sln</i> deletion does not rescue SERCA function in <i>Pln</i>^OE mice.

<p>(A) Western blot analyses showing successful removal of the 8-fold upregulation in SLN protein (n = 6 per genotype). Ponceau staining ensured equal loading. (B) <i>Pln</i>^OE and <i>Pln</i>^OE/<i>Sln</i>^KO soleus muscles displayed similar levels of monomeric (m) and pentameric (p) PLN overexpression compared with WT (n = 6 per genotype). Values were normalized to actin. (C) Rates of Ca²⁺ uptake were similarly reduced in the <i>Pln</i>^OE and <i>Pln</i>^OE/<i>Sln</i>^KO soleus muscles compared with WT (n = 6–7, per genotype). (D) Representative SERCA activity-<i>p</i>Ca curves measured in soleus homogenates (n = 6–7, per genotype). (E) SERCA’s apparent affinity for Ca²⁺, presented as <i>p</i>Ca₅₀, was similarly reduced in the <i>Pln</i>^OE and <i>Pln</i>^OE/<i>Sln</i>^KO soleus muscles compared with WT (n = 6–7, per genotype). <i>p</i>Ca₅₀ is the negative logarithm of the Ca²⁺ concentration required to attain half-maximal SERCA activity rate. *Significantly different from WT using a Student’s t-test (A), and a one-way ANOVA with a Tukey’s post-hoc (B, C and E), <i>p</i> ≤ 0.05. All values are means ± SEM.</p

<i>Sln</i> deletion does not augment proteolytic activity and autophagic signaling in response to <i>Pln</i> overexpression.

<p>Calpain (A), caspase-3 (B), and 20S proteasome activity assays (C). Enhanced autophagy revealed by increased cathepsin activity (D), decreased p62 levels (E) and increased LC3-II/I content (F) in <i>Pln</i>^OE mice is attenuated in <i>Pln</i>^OE/<i>Sln</i>^KO mice. *Significantly different from WT, ^†significantly different from <i>Pln</i>^OE using a one-way ANOVA and a Tukey’s post-hoc test, <i>p</i> ≤ 0.05. For proteolytic measures, n = 6–7; for p62, LC3-I and–II content, n = 11–12 and optical densities were normalized to actin. All values are means ± SEM.</p

Body weight, daily activity and daily food intake measures from WT, <i>Pln</i>^OE, and <i>Pln</i>^OE/<i>Sln</i>^KO mice.

<p>Body weight, daily activity and daily food intake measures from WT, <i>Pln</i>^OE, and <i>Pln</i>^OE/<i>Sln</i>^KO mice.</p

Without SLN upregulation, <i>Pln</i>^OE soleus muscles are smaller and weaker.

<p>(A) soleus:body weight ratios (mg/g) across genotype (n = 18–23 per genotype). (B) Total myofiber count in soleus muscle sections counted after immunofluorescent staining (n = 6 per genotype). (C) Mass-specific force-frequency curve analysis in isolated soleus muscles (n = 6–7 per genotype). Rates of relaxation (-dF/dt, D) and force development (+dF/dt, E) measured for a single twitch (n = 6–7 per genotype). For (A, B, D and E), *significantly different from WT; ^†significantly different from <i>Pln</i>^OE, using a one-way ANOVA <i>p</i> ≤ 0.05. For C, *significantly different from <i>Pln</i>^OE/<i>Sln</i>^KO; ^†significantly different from <i>Pln</i>^OE, using a two-way ANOVA <i>p</i> ≤ 0.05. All values are means ± SEM.</p