Does Sarcolipin Ablation Alter Deflazacort Treatment Effects in mdx Mice?

Duchenne Muscular Dystrophy (DMD) and the murine model mdx are degenerative diseases that are characterized by the absence of the protein dystrophin causing membrane instability, calcium (Ca2+) influx, and progressive muscle wasting. The sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) is the enzyme responsible for maintaining low cytosolic Ca2+ and is dysfunctional in mdx mice, contributing to elevations in cytosolic Ca2+. Sarcolipin (SLN) is an endogenous protein regulator of SERCA that reduces SERCA activity by lowering the apparent Ca2+ affinity of SERCA. SLN is highly upregulated in mdx muscle, yet when ablated from mdx mice a worsened phenotype is observed, possibly due to blunted calcineurin/NFAT signaling. Glucocorticoid steroids are the primary therapy for DMD patients and combat the dystrophic phenotype in mdx mice. Glucocorticoid steroids have been shown to stimulate calcineurin signaling, which results in the upregulation of utrophin, the homologue of dystrophin. Glucocorticoids also increase the expression of SLN, which may function to regulate calcineurin activity. The aim of this study was to determine whether SLN plays a role in the positive treatment effects of glucocorticoid steroids in mdx mice. It was hypothesized that glucocorticoid treatment would increase SLN expression, stimulate calcineurin activity and increase utrophin expression in muscles from mdx mice but not in muscles from mdx mice lacking SLN (i.e. mdx/Sln-/-). Here, four week old mdx and mdx/Sln-/- mice were injected with either the glucocorticoid, deflazacort (DFZ), or vehicle (VEH) alone for 7 days. Following treatment, soleus (SOL) and diaphragm (DIA) muscles were collected for biochemical and histological analyses. Protein content of SLN, utrophin, calcineurin, SERCA isoforms and nuclear factor activation T cell (NFAT), a transcription factor dephosphorylated by calcineurin, was determined using western blotting. Immunofluorescent/histological techniques were used to assess centralized nuclei, collagen infiltration, myosin heavy chain isoform, cross sectional area (CSA), and fibre size variability, using the variance coefficient of minimal Feret’s diameter. Activation of the calcineurin/NFAT signaling pathway was also assessed using co-localization of NFATc1 and DAPI, which stains nuclear DNA. DFZ treatment improved several markers of the dystrophic phenotype in both the SOL and DIA, including centrally nucleated fibres and collagen infiltration. There were some muscle-specific differences in fibre type distribution between genotypes and treatments but these were mostly minor differences in hybrid fibres. CSA was not affected in the majority of fibre types by DFZ or genotype. Unexpectedly, variability in fibre size was not improved by DFZ treatment but was actually increased in DFZ treated mdx/Sln-/- DIA compared with VEH. DFZ increased NFATc1 and DAPI co-localization in both mdx and mdx/Sln-/- mice in the SOL only. However, calcineurin content and utrophin in both SOL and DIA were not different between any groups. SERCA1a and SERCA2a content in SOL and DIA were not different between DFZ and VEH treated mice, but SERCA1a was lower in the DIA of mdx/Sln-/- mice compared to mdx mice. DFZ treatment increased SLN content in mdx SOL but not in DIA. In summary, 7 days of DFZ treatment improved some histological features of DMD in both mdx and mdx/Sln-/- mice in the SOL, which was associated with increased NFATc1 nuclear translocation, suggesting increased activation of the calcineurin/NFAT pathway. Despite histological improvements in the DIA with DFZ treatment, the calcineurin/NFAT pathway appeared not to be active in that tissue relative to VEH. Finally, contrary to the main hypothesis, DFZ effects were similar in mdx and mdx/Sln-/- mice, suggesting that SLN does not play a role in mediating the positive effects of DFZ

Aetiology and use of antibiotics in pregnancy-related infections: results of the WHO Global Maternal Sepsis Study (GLOSS), 1-week inception cohort

Background Pregnancy-related infections are important contributors to maternal sepsis and mortality. We aimed to describe clinical, microbiological characteristics and use of antibiotics by source of infection and country income, among hospitalized women with suspected or confirmed pregnancy-related infections. Methods We used data from WHO Global Maternal Sepsis Study (GLOSS) on maternal infections in hospitalized women, in 52 low-middle- and high-income countries conducted between November 28th and December 4th, 2017, to describe the frequencies and medians of maternal demographic, obstetric, and clinical characteristics and outcomes, methods of infection diagnosis and causative pathogens, of single source pregnancy-related infection, other than breast, and initial use of therapeutic antibiotics. We included 1456 women. Results We found infections of the genital (n = 745/1456, 51.2%) and the urinary tracts (UTI) (n = 531/1456, 36.5%) to be the most frequent. UTI (n = 339/531, 63.8%) and post-caesarean skin and soft tissue infections (SSTI) (n = 99/180, 55.0%) were the sources with more culture samples taken and microbiological confirmations. Escherichia coli was the major uropathogen (n = 103/118, 87.3%) and Staphylococcus aureus (n = 21/44, 47.7%) was the commonest pathogen in SSTI. For 13.1% (n = 191) of women, antibiotics were not prescribed on the same day of infection suspicion. Cephalosporins (n = 283/531, 53.3%) were the commonest antibiotic class prescribed for UTI, while metronidazole (n = 303/925, 32.8%) was the most prescribed for all other sources. Ceftriaxone with metronidazole was the commonest combination for the genital tract (n = 98/745, 13.2%) and SSTI (n = 22/180, 12.2%). Metronidazole (n = 137/235, 58.3%) was the most prescribed antibiotic in low-income countries while cephalosporins and co-amoxiclav (n = 129/186, 69.4%) were more commonly prescribed in high-income countries. Conclusions Differences in antibiotics used across countries could be due to availability, local guidelines, prescribing culture, cost, and access to microbiology laboratory, despite having found similar sources and pathogens as previous studies. Better dissemination of recommendations in line with antimicrobial stewardship programmes might improve antibiotic prescription

Mitochondrial bioenergetic dysfunction in the D2.mdx model of Duchenne muscular dystrophy is associated with microtubule disorganization in skeletal muscle.

In Duchenne muscular dystrophy, a lack of dystrophin leads to extensive muscle weakness and atrophy that is linked to cellular metabolic dysfunction and oxidative stress. This dystrophinopathy results in a loss of tethering between microtubules and the sarcolemma. Microtubules are also believed to regulate mitochondrial bioenergetics potentially by binding the outer mitochondrial membrane voltage dependent anion channel (VDAC) and influencing permeability to ADP/ATP cycling. The objective of this investigation was to determine if a lack of dystrophin causes microtubule disorganization concurrent with mitochondrial dysfunction in skeletal muscle, and whether this relationship is linked to altered binding of tubulin to VDAC. In extensor digitorum longus (EDL) muscle from 4-week old D2.mdx mice, microtubule disorganization was observed when probing for α-tubulin. This cytoskeletal disorder was associated with a reduced ability of ADP to stimulate respiration and attenuate H2O2 emission relative to wildtype controls. However, this was not associated with altered α-tubulin-VDAC2 interactions. These findings reveal that microtubule disorganization in dystrophin-deficient EDL is associated with impaired ADP control of mitochondrial bioenergetics, and suggests that mechanisms alternative to α-tubulin's regulation of VDAC2 should be examined to understand how cytoskeletal disruption in the absence of dystrophin may cause metabolic dysfunctions in skeletal muscle

Aetiology and use of antibiotics in pregnancy-related infections: results of the WHO global maternal sepsis study (GLOSS), 1-week inception cohort

Background: Pregnancy-related infections are important contributors to maternal sepsis and mortality. We aimed to describe clinical, microbiological characteristics and use of antibiotics by source of infection and country income, among hospitalized women with suspected or confirmed pregnancy-related infections.Methods: We used data from WHO Global Maternal Sepsis Study (GLOSS) on maternal infections in hospitalized women, in 52 low-middle- and high-income countries conducted between November 28th and December 4th, 2017, to describe the frequencies and medians of maternal demographic, obstetric, and clinical characteristics and outcomes, methods of infection diagnosis and causative pathogens, of single source pregnancy-related infection, other than breast, and initial use of therapeutic antibiotics. We included 1456 women.Results: We found infections of the genital (n = 745/1456, 51.2%) and the urinary tracts (UTI) (n = 531/1456, 36.5%) to be the most frequent. UTI (n = 339/531, 63.8%) and post-caesarean skin and soft tissue infections (SSTI) (n = 99/180, 55.0%) were the sources with more culture samples taken and microbiological confirmations. Escherichia coli was the major uropathogen (n = 103/118, 87.3%) and Staphylococcus aureus (n = 21/44, 47.7%) was the commonest pathogen in SSTI. For 13.1% (n = 191) of women, antibiotics were not prescribed on the same day of infection suspicion. Cephalosporins (n = 283/531, 53.3%) were the commonest antibiotic class prescribed for UTI, while metronidazole (n = 303/925, 32.8%) was the most prescribed for all other sources. Ceftriaxone with metronidazole was the commonest combination for the genital tract (n = 98/745, 13.2%) and SSTI (n = 22/180, 12.2%). Metronidazole (n = 137/235, 58.3%) was the most prescribed antibiotic in low-income countries while cephalosporins and co-amoxiclav (n = 129/186, 69.4%) were more commonly prescribed in high-income countries.Conclusions: Differences in antibiotics used across countries could be due to availability, local guidelines, prescribing culture, cost, and access to microbiology laboratory, despite having found similar sources and pathogens as previous studies. Better dissemination of recommendations in line with antimicrobial stewardship programmes might improve antibiotic prescription

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

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

Phospholamban overexpression in mice causes a centronuclear myopathy-like phenotype

Centronuclear myopathy (CNM) is a congenital myopathy that is histopathologically characterized by centrally located nuclei, central aggregation of oxidative activity, and type I fiber predominance and hypotrophy. Here, we obtained commercially available mice overexpressing phospholamban (PlnOE), a well-known inhibitor of sarco(endo)plasmic reticulum Ca2+-ATPases (SERCAs), in their slow-twitch type I skeletal muscle fibers to determine the effects on SERCA function. As expected with a 6- to 7-fold overexpression of phospholamban, SERCA dysfunction was evident in PlnOE muscles, with marked reductions in rates of Ca2+ uptake, maximal ATPase activity and the apparent affinity of SERCA for Ca2+. However, our most significant discovery was that the soleus and gluteus minimus muscles from the PlnOE mice displayed overt signs of myopathy: they histopathologically resembled human CNM, with centrally located nuclei, central aggregation of oxidative activity, type I fiber predominance and hypotrophy, progressive fibrosis and muscle weakness. This phenotype is associated with significant upregulation of muscle sarcolipin and dynamin 2, increased Ca2+-activated proteolysis, oxidative stress and protein nitrosylation. Moreover, in our assessment of muscle biopsies from three human CNM patients, we found a significant 53% reduction in SERCA activity and increases in both total and monomeric PLN content compared with five healthy subjects, thereby justifying future studies with more CNM patients. Altogether, our results suggest that the commercially available PlnOE mouse phenotypically resembles human CNM and could be used as a model to test potential mechanisms and therapeutic strategies. To date, there is no cure for CNM and our results suggest that targeting SERCA function, which has already been shown to be an effective therapeutic target for murine muscular dystrophy and human cardiomyopathy, might represent a novel therapeutic strategy to combat CNM

<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