Orai1 Mediates Exacerbated Ca2+ Entry in Dystrophic Skeletal Muscle

There is substantial evidence indicating that disruption of Ca(2+) homeostasis and activation of cytosolic proteases play a key role in the pathogenesis and progression of Duchenne Muscular Dystrophy (DMD). However, the exact nature of the Ca(2+) deregulation and the Ca(2+) signaling pathways that are altered in dystrophic muscles have not yet been resolved. Here we examined the contribution of the store-operated Ca(2+) entry (SOCE) for the pathogenesis of DMD. RT-PCR and Western blot found that the expression level of Orai1, the pore-forming unit of SOCE, was significantly elevated in the dystrophic muscles, while parallel increases in SOCE activity and SR Ca(2+) storage were detected in adult mdx muscles using Fura-2 fluorescence measurements. High-efficient shRNA probes against Orai1 were delivered into the flexor digitorum brevis muscle in live mice and knockdown of Orai1 eliminated the differences in SOCE activity and SR Ca(2+) storage between the mdx and wild type muscle fibers. SOCE activity was repressed by intraperitoneal injection of BTP-2, an Orai1 inhibitor, and cytosolic calpain1 activity in single muscle fibers was measured by a membrane-permeable calpain substrate. We found that BTP-2 injection for 2 weeks significantly reduced the cytosolic calpain1 activity in mdx muscle fibers. Additionally, ultrastructural changes were observed by EM as an increase in the number of triad junctions was identified in dystrophic muscles. Compensatory changes in protein levels of SERCA1, TRP and NCX3 appeared in the mdx muscles, suggesting that comprehensive adaptations occur following altered Ca(2+) homeostasis in mdx muscles. Our data indicates that upregulation of the Orai1-mediated SOCE pathway and an overloaded SR Ca(2+) store contributes to the disrupted Ca(2+) homeostasis in mdx muscles and is linked to elevated proteolytic activity, suggesting that targeting Orai1 activity may be a promising therapeutic approach for the prevention and treatment of muscular dystrophy

Repurposing of Meropenem and Nadifloxacin for Treatment of Burn Patients?

The escalating number of multidrug resistant pathogens has demanded the swift development of new and potent antibiotics (ref. 2). Metallo-[beta]-lactamases (MBLs) continue to evolve, rendering the latest generation of carbapenem antibiotics useless (ref. 8). SPM-1, a recently discovered MBL, was isolated from a juvenile leukemia patient residing in a hospital in San Palo, Brazil just prior to the patient succumbing to septicemia brought on by Pseudomonas aeruginosa expressing SPM-1 (ref. 8). Screening of the Johns Hopkins Compound library of 1,514 FDA or FAD approved drugs (ref. 1) identified a novel SPM-1 inhibitor that is synergistically compatible with meropenem. Using clinically achievable concentrations, meropenem coupled with nadifloxacin inhibits Pseudomonas aeruginosa expressing SPM-1. This shotgun approach to new drug discovery provided a prompt solution to the grave problem of antibiotic resistant pathogens that are thriving in hospitals today

Up-regulation of Orai1 in <i>mdx</i> muscle.

<p>(<b>A</b>) mRNA expression levels of Orai1 and STIM1 in gastrocnemius muscles from <i>wt</i> C57BL/10ScSnJ and dystrophic C57BL/10ScSn-Dmd<i>^mdx</i>/J mice were detected by real-time PCR. Relative mRNA copy numbers were shown, n = 6–8 for Orai1 and n = 4 for STIM1, ^*<i>P</i><0.05. (<b>B</b>) Western blot showed upregulation of Orai1 protein (∼50 kD) in <i>wt</i> and <i>mdx</i> FDB muscles. The observed molecular size of Orai1 protein was higher than the predicted molecular weight of 33 kDa, possibly due to post-translational modification, splice variations or changes in relative charges of the amino acid of the protein. Sarcomeric α-actin (42 kD) was used as a loading control and for normalization of densitometry, n = 6∼7, ^*<i>P</i><0.05.</p

Effective knockdown of endogenous Orai1 gene in both <i>wt</i> and <i>mdx</i> FDB fibers by shRNA probe.

<p>(<b>A</b>) Superimposed transmission light and red fluorescent images (100x) of Hela cells after 24 h transfection with shOrai1 with a RFP marker and mouse full length Orai1 with a myc tag (mOrai1-myc). (<b>B</b>) Western blot showing effective knockdown of the exogenous mOrai1 gene by shOrai1 probes (sh1∼3) No. 2 and No. 3. β-actin was used as a loading control. (<b>C</b>) Fluorescence image (100x) showing the successful transfection of FDB muscle 2 weeks after electroporation of shOrai1 probe. (<b>D</b>) Single fiber western blots confirmed the effectiveness of shOrai1 in knocking down the endogenous mOrai1 in <i>wt</i> mice (upper panel) and <i>mdx</i> mice (lower panel). Pooled extracts from 15 individual FDB fibers were loaded per lane. The exposure used to generate these images was adjusted to produce a robust signal that could be used to assess the level of Orai1 knockdown for both <i>wt</i> and <i>mdx</i> mice. (<b>E</b>) Densitometry of Orai1 Western blot in wild type muscle transfected with control or shOrai1 vectors using NIH image J, n = 2. (<b>F</b>) Densitometry of Orai1 Western blot in mdx muscle transfected with control or shOrai1 vector, n = 3, *<i>P</i><0.10.</p

Compensatory change in protein expression of other Ca²⁺ shuttling pathways in <i>mdx</i> muscles.

<p>(<b>A</b>) Absence of the 427 kDa dystrophin in <i>mdx</i> muscles was confirmed and expression levels of STIM1 and SERCA1 in FDB muscles from the <i>wt</i> C57BL/10ScSnJ and dystrophic C57BL/10ScSn-Dmd<i>^mdx</i>/J mice were tested by western blot. Sarcomeric a-actin was used as a loading control. (<b>B</b>) In a separate study, the levels of TRPC3/6 and NCX3 were tested in <i>wt</i> and <i>mdx</i> muscles. (<b>C</b>) Densitometry of detected protein expression relative to α-actin, n = 2 for STIM1 and SERCA1 and n = 3 for TrpC3/C6 and NCX, *<i>P</i><0.10.</p

SOCE inhibitor reduces calpain activation in dystrophic fibers.

<p>(<b>A</b>) Confocal images of FDB fibers from DMSO-injected <i>mdx</i> mice and BTP2 injected <i>mdx</i> mice at T₀ (addition of 50 µM fluorogenic, membrane-permeable peptide, Suc-LLVY-AMC) and T₁ (25 min later to allow for penetration of Suc-LLVY-AMC into FDB fiber and cleavage by cytosolic calpain) at excitation wavelength of 360 nm and emission wavelength of 460 nm. The lower transmission panel is to show that only healthy muscle fibers with clear striation pattern were chosen for the experiment; (<b>B</b>) Statistical analysis of changes in the fluorescence signals (ΔF) generated by Suc-LLVY-AMC cleavage in control <i>mdx</i> fiber (open bar) and BTP2-treated <i>mdx</i> fiber (hatched bar), n = 8 (BTP2) and 14 (DMSO), ^*<i>P</i><0.05.</p

Elevation of Orai1-mediated SOCE activity and SR Ca²⁺ store overload in adult <i>mdx</i> muscles.

<p>(<b>A</b>) Representative trace of Mn²⁺ quenching of Fura-2 fluorescence at 360 nm (F₃₆₀) wavelength. Lines and arrows designate perfusion of muscle fiber by 20 mM caffeine plus 5 uM ryanodine, MnCl₂ and TritonX-100. Upper panel: black trace is FDB fiber from <i>wt</i> mice transfected with empty vector (control WT) and grey trace is with shOrai1 (Orai1KD WT); lower panel: black trace is FDB fiber from <i>mdx</i> mice transfected with empty vector (Control <i>mdx</i>) and grey trace is with Orai1 (Orai1KD <i>mdx</i>). (<b>B</b>) Statistical summarization of the data in (A), n = 11–19, ^*<i>P</i><0.05 compared to Control WT; ^#<i>P</i><0.05 compared to Control <i>mdx</i>. (<b>C</b>) Statistical results of resting intracellular Ca²⁺ levels in Control WT (open bar), Orai1KD WT (black bar), Control <i>mdx</i> (hatched bar) and Orai1KD <i>mdx</i> (black bar). (<b>D</b>) Statistical results of caffeine-sensitive SR Ca²⁺ store in the four groups. n = 11–19, ^*<i>P</i><0.05 compared to Control WT; ^#<i>P</i><0.05 compared to Control <i>mdx</i>.</p

Changes in muscle ultrastructure after Orai1 knockdown.

<p>Longitudinal section of FDB muscles under transmission EM of the (<b>A</b>) <i>wt</i> muscle transfected with control vector. Scale bar is 200 nm. Black arrows designate triads at A-I junctions and white arrows designate triad-defect A-I junctions; (<b>B</b>) <i>mdx</i> muscle transfected with control vector. Red arrows designate the dense triads at A-I junctions; (<b>C</b>) abnormal features observed in muscles with Orai1 knockdown. Left: disoriented triad junctional structure (white arrows); right: thick and rough reticulation as compared to the fine SR network structure in <i>wt</i> (A) and <i>mdx</i> control (B) muscle; (<b>D</b>) Statistical results summarize the percentage of triad-containing A-I junctions in muscle bundles from <i>wt</i> control, <i>wt</i> Orai1 knockdown, <i>mdx</i> control and <i>mdx</i> Orai1 knockdown. Data are mean ± S.E.M. *<i>P</i><0.05 compared to <i>wt</i> control and ^#<i>P</i><0.05 compared to <i>mdx</i> control.</p