Additional file 2: Figure S2. of The Binding Sites of miR-619-5p in the mRNAs of Human and Orthologous Genes

Variation of nucleotide sequences of mRNA region with miR-619-5p binding sites of genes from GK5 to HM13 (Conservative binding sites are in bold) (PDF 106 kb

Role of autophagy, SQSTM1, SH3GLB1, and TRIM63 in the turnover of nicotinic acetylcholine receptors

<p>Removal of ubiquitinated targets by autophagosomes can be mediated by receptor molecules, like SQSTM1, in a mechanism referred to as selective autophagy. While cytoplasmic protein aggregates, mitochondria, and bacteria are the best-known targets of selective autophagy, their role in the turnover of membrane receptors is scarce. We here showed that fasting-induced wasting of skeletal muscle involves remodeling of the neuromuscular junction (NMJ) by increasing the turnover of muscle-type CHRN (cholinergic receptor, nicotinic/nicotinic acetylcholine receptor) in a TRIM63-dependent manner. Notably, this process implied enhanced production of endo/lysosomal carriers of CHRN, which also contained the membrane remodeler SH3GLB1, the E3 ubiquitin ligase, TRIM63, and the selective autophagy receptor SQSTM1. Furthermore, these vesicles were surrounded by the autophagic marker MAP1LC3A in an ATG7-dependent fashion, and some of them were also positive for the lysosomal marker, LAMP1. While the amount of vesicles containing endocytosed CHRN strongly augmented in the absence of ATG7 as well as upon denervation as a model for long-term atrophy, denervation-induced increase in autophagic CHRN vesicles was completely blunted in the absence of TRIM63. On a similar note, in <i>trim63^−/−</i> mice denervation-induced upregulation of SQSTM1 and LC3-II was abolished and endogenous SQSTM1 did not colocalize with CHRN vesicles as it did in the wild type. SQSTM1 and LC3-II coprecipitated with surface-labeled/endocytosed CHRN and SQSTM1 overexpression significantly induced CHRN vesicle formation. Taken together, our data suggested that selective autophagy regulates the basal and atrophy-induced turnover of the pentameric transmembrane protein, CHRN, and that TRIM63, together with SH3GLB1 and SQSTM1 regulate this process.</p

Fast Skeletal Muscle Troponin Activation Increases Force of Mouse Fast Skeletal Muscle and Ameliorates Weakness Due to Nebulin-Deficiency

<div><p>The effect of the fast skeletal muscle troponin activator, CK-2066260, on calcium-induced force development was studied in skinned fast skeletal muscle fibers from wildtype (WT) and nebulin deficient (NEB KO) mice. Nebulin is a sarcomeric protein that when absent (NEB KO mouse) or present at low levels (nemaline myopathy (NM) patients with NEB mutations) causes muscle weakness. We studied the effect of fast skeletal troponin activation on WT muscle and tested whether it might be a therapeutic mechanism to increase muscle strength in nebulin deficient muscle. We measured tension–pCa relations with and without added CK-2066260. Maximal active tension in NEB KO tibialis cranialis fibers in the absence of CK-2066260 was ∼60% less than in WT fibers, consistent with earlier work. CK-2066260 shifted the tension-calcium relationship leftwards, with the largest relative increase (up to 8-fold) at low to intermediate calcium levels. This was a general effect that was present in both WT and NEB KO fiber bundles. At pCa levels above ∼6.0 (i.e., calcium concentrations <1 µM), CK-2066260 increased tension of NEB KO fibers to beyond that of WT fibers. Crossbridge cycling kinetics were studied by measuring <i>k_tr</i> (rate constant of force redevelopment following a rapid shortening/restretch). CK-2066260 greatly increased <i>k_tr</i> at submaximal activation levels in both WT and NEB KO fiber bundles. We also studied the sarcomere length (SL) dependence of the CK-2066260 effect (SL 2.1 µm and 2.6 µm) and found that in the NEB KO fibers, CK-2066260 had a larger effect on calcium sensitivity at the long SL. We conclude that fast skeletal muscle troponin activation increases force at submaximal activation in both wildtype and NEB KO fiber bundles and, importantly, that this troponin activation is a potential therapeutic mechanism for increasing force in NM and other skeletal muscle diseases with loss of muscle strength.</p> </div

Effect of 10 µM CK-2066260 on tension-pCa curves at short (2.1 µm) and long SL (2.6 µm).

<p>Result in WT in A and in NEB KO fibers in B. Increasing SL slightly shifts the curves left-ward (i.e., calcium sensitivity is increased) and CK results in an additional large shift. Results from six WT and six KO mice.</p

Tension–pCa relations in WT and NEB KO skinned TC muscle fibers.

<p>A) Average active tension-pCa curves in WT (open symbols, dashed) and KO (closed symbols, solid) mice with 0 (blue), 5 µM (green) and 10 µM (red) CK-2066260 at SL = 2.1 µm. Curves in CK were shifted to the left in both WT and KO, i.e. calcium sensitivity is increased. Note that at pCa>6.0, CK-2066260 increased active tension of NEB-KO muscle beyond that of WT. B) Relative active tension increase (plotted on a log scale) in 5 and 10 µM of CK over 0 µM of CK at various pCa levels. Regardless of genotype, active tensions were significantly increased from ∼800% at the pCa 6.4 to ∼10% at pCa 4.5. Results from five WT and five KO mice.</p

Effect of CK 2066260 on the rate constant of force redevelopment (<i>k_tr</i>).

<p>A) Examples of <i>k_tr</i> experiments on WT and NEB KO fiber bundles activated at pCa 5.95 before (control) and after the addition of 10 µM CK-2066260. Shown is the tension recovery phase following the re-stretch (see Methods for details). In the presence of 10 µM CK-2066260 (darker traces) tension recovery is faster than in absence of CK-2066260. B) and C) k<i>_tr</i> results of WT (B) and NEB KO fibers (C). Addition of 10 µM CK-2066260 increases <i>k_tr</i> in both WT and NEB-KO fibers. A two-way ANOVA reveals in WT bundles significant effects of pCa and CK-2066260 on <i>k_tr</i> with a significant interaction between pCa and CK (p<0.001). The same is the case for the KO data, except that the p-value of the interaction term is p = 0.02. D) Change in <i>k_tr</i> in CK 2066260 (as percentage of control values). Results from six WT and six KO mice.</p

SL-induced ΔpCa₅₀ at 0 and 10 µM CK-2066260 and CK 2066260-induced ΔpCa₅₀ at SL 2.1 µm and SL 2.6 µm.

<p>A) Effect of increasing SL from 2.1 to 2.6 µm on calcium sensitivity (SL-induced ΔpCa₅₀) without and with 10 µM CK 2066260. In KO fibers CK-2066260 increased the SL-induced ΔpCa₅₀ (#, p<0.05). B) CK-2066260 (10 µM) induced ΔpCa₅₀ at SL 2.1 µm and 2.6 µm. Only in KO fibers CK 2066260-induced ΔpCa₅₀ was significantly greater at SL 2.6 µm compared to 2.1 µm (†, p<0.05). Furthermore, at SL 2.6 µm CK 2066260-induced ΔpCa₅₀ in KO fibers was significantly greater than in WT (*, p<0.05). Results from six WT and six KO mice.</p

T_max (tension at pCa 4.5); calcium sensitivity (pCa₅₀) and Hill coefficient (n_H) of WT and KO in 0, 5, and 10 µM CK-2066260.

<p>Results are mean ± SEM from 5 WT and 5 KO mice, except for the last 4 rows that are from 6 WT and 6 KO mice.</p>*<p>comparison WT vs. KO;</p>#<p>comparison versus 0 µM CK-2066260;</p>√<p>comparison versus 5 µM CK-2066260; single symbol p<0.05, double symbol p<0.01. All measurements were at a sarcomere length (SL) of 2.1 µm, except the bottom 4 rows that were at 2.6 µm.</p

Experimental Protocol and effect of CK-2066260 on active tension at submaximal activation.

<p>A) Skinned skeletal muscle fibers (<i>TC</i> muscle) were stretched, held for 7 min, and were then released. During the hold phase, the muscle was exposed to various pCa activating solutions, and was then relaxed again. B) Effect of CK-2066260 on active tension at submaximal activation level (pCa = 6.25). Active tensions at a range of CK-2066260 concentrations (black symbols). The maximal active tension (pCa 4.5) at 0 µM CK-2066260 is shown to the right in red.</p

A-SAA serum levels and <i>IL-6</i> and <i>TNF-α</i> expression in skeletal muscle of critically ill patients.

<p>(A) Serum levels of acute phase SAA (A-SAA) measured by ELISA in healthy controls (n = 6), critically ill patients (ICUs, n = 30), non-CIM (n = 19) and CIM (n = 11) patients. Serum samples were obtained at days 2 to 3 after ICU admission. **<i>P</i><0.01, *<i>P</i><0.05. (B) RT-PCR analyses of <i>IL-6</i> and <i>TNF-α</i> expressions in skeletal muscle from critically ill patients at early (day 5) and late (day 15) time points. <i>Glyceraldehyde-3 phosphate dehydrogenase</i> (<i>GAPDH</i>) expression was used as reference. (C) RT-PCR analyses of <i>IL-6</i> and <i>TNF-α</i> expression at early and late time points in CIM and non-CIM patients. Data are presented as box plots showing median, 25^th and 75^th percentiles. Wilcoxon tests were performed between early and late biopsy specimens and Mann-Whitney tests for the respective time points and controls; ***<i>P</i><0.001, **<i>P</i><0.01, *<i>P</i><0.05, or n.s. (not statistically significant).</p