Long noncoding RNA H19X is a key mediator of TGF-beta-driven fibrosis

TGFβ is a master regulator of fibrosis, driving the differentiation of fibroblasts into apoptosis resistant myofibroblasts and sustaining the production of extracellular matrix (ECM) components. Here, we identify the nuclear lncRNA H19X as a master regulator of TGFβ-driven tissue fibrosis. H19X was consistently upregulated in a wide variety of human fibrotic tissues and diseases and was strongly induced by TGFβ, particularly in fibroblasts and fibroblast-related cells. Functional experiments following H19X silencing revealed that H19X is an obligatory factor for the TGFβ-induced ECM synthesis as well as differentiation and survival of ECM-producing myofibroblasts. We showed that H19X regulates DDIT4L gene expression, specifically interacting with a region upstream of DDIT4L gene and changing the chromatin accessibility of a DDIT4L enhancer. These events resulted in transcriptional repression of DDIT4L and, in turn, in increased collagen expression and fibrosis. Our results shed light on key effectors of the TGFβ-induced ECM remodeling and fibrosis

The Effects of Low Levels of Dystrophin on Mouse Muscle Function and Pathology

Duchenne muscular dystrophy (DMD) is a severe progressive muscular disorder caused by reading frame disrupting mutations in the DMD gene, preventing the synthesis of functional dystrophin. As dystrophin provides muscle fiber stability during contractions, dystrophin negative fibers are prone to exercise-induced damage. Upon exhaustion of the regenerative capacity, fibers will be replaced by fibrotic and fat tissue resulting in a progressive loss of function eventually leading to death in the early thirties. With several promising approaches for the treatment of DMD aiming at dystrophin restoration in clinical trials, there is an increasing need to determine more precisely which dystrophin levels are sufficient to restore muscle fiber integrity, protect against muscle damage and improve muscle function. To address this we generated a new mouse model (mdx-Xist Dhs) with varying, low dystrophin levels (3–47%, mean 22.7%, stdev 12.1, n = 24) due to skewed X-inactivation. Longitudinal sections revealed that within individual fibers, some nuclei did and some did not express dystrophin, resulting in a random, mosaic pattern of dystrophin expression within fibers. Mdx-Xist Dhs, mdx and wild type females underwent a 12 week functional test regime consisting of different tests to assess muscle function at base line, or after chronic treadmill running exercise. Overall, mdx-Xist Dhs mice with 3–14 % dystrophin outperformed mdx mice in the functional tests. Improved histopathology was observed in mice with 15–29 % dystrophin and these levels also resulted in normalized expression of pro-inflammatory biomarker genes, while for other parameters.30 % of dystrophin was needed

Expression of several genes involved in disease pathology.

<p>A and B. For most biomarkers a clear dystrophin level dependent restoration of gene expression levels was observed in the <i>mdx-Xist</i>^Δhs mice, where intermediate dystrophin levels resulted in low expression of genes involved in disease pathology. For some genes, dystrophin levels <15% were enough to reduce gene expression while for other genes >30% was necessary. C. In the heart, even dystrophin levels of <15% decreased expression of fibrotic biomarkers like <i>Timp-1</i>. Since the mice were young, no difference in expression levels in heart function was observed, except for <i>Nppa</i>. Double asterisks indicate a <i>P</i><0.01.</p

<i>Mdx-Xist</i>^Δhs mice.

<p>A. To breed mice with low dystrophin levels, female <i>Xist</i>^Δhs mice, carrying a mutation in the <i>Xist</i> promoter which coordinates X-inactivation, were crossed with dystrophin negative <i>mdx</i> males. During embryogenesis, the maternal X-chromosome encoding a functional dystrophin gene will be preferentially (60–90%) inactivated as a result of the mutated <i>Xist</i> promoter. The <i>Xist</i>^Δhs mice were a kind gift from N. Brockdorff (MRC Clinical Sciences Center, London, UK, current affiliation Department of Biochemistry, University of Oxford, UK). B. Picture of a representative Western blot. The percentage of dystrophin was determined for the quadriceps of all <i>mdx-Xist</i>^Δhs mice by Western blot (2–9 technical replicates per mouse). The percentage of individual mice was determined using a concentration curve made from wild type samples. Myosin was used as a loading control. C. Skewed X-inactivation resulted in dystrophin levels of 3–47% (mean 22.7, stdev 12.1, <i>n</i> = 24) (as determined by Western blot) in the female <i>mdx-Xist</i>^Δhs offspring. Each bar represents the dystrophin level of an individual mouse. The dystrophin levels of the individual mice belonging to the three dystrophin groups can be appreciated from this graph. D. Dystrophin levels determined by Western blot and manual counting of dystrophin positive fibers demonstrate a strong correlation (R = 0.97). E. Longitudinal sections of a quadriceps stained with dystrophin (green) and spectrin (red). From the pictures it can be appreciated that dystrophin expression is not homogeneously expressed across the fiber but rather confined to certain nuclear domains.</p

Exercise induced histopathology and biomarker gene expression.

<p>A. Wild type mice were less severely affected than <i>mdx</i> and <i>mdx-Xist</i>^Δhs mice. Due to high variation between individual <i>mdx</i> mice, the difference between <i>mdx</i> and wild type mice was not significant. <i>Mdx-Xist</i>^Δhs mice had slightly less fibrotic tissue than <i>mdx</i> mice. B. No difference in expression of genes involved in disease pathology was found between <i>mdx</i> and <i>mdx-Xist</i>^Δhs mice. Both mouse strains did differ significantly from wild type mice, of which the expression levels were low. Single and double asterisks indicate a <i>P</i><0.05 and <i>P</i><0.01, respectively. # Indicates a significant difference from all other groups, average dystrophin levels of <i>mdx-Xist</i>^Δhs mice was 21% (2%–45% median 25.8).</p

Functional performance measured directly after treadmill exercise.

<p>A. Longest hanging time with the two limb hanging test was achieved by wild type and <i>mdx-Xist</i>^Δhs mice which both performed significantly (<i>P</i><0.001) better than <i>mdx</i> mice. B. Fore limb grip strength of <i>mdx-Xist</i>^Δhs mice was significantly (<i>P</i><0.001) better than that of <i>mdx</i> mice. C. Wild type mice outperformed <i>mdx</i> and <i>mdx-Xist</i>^Δhs mice on the rotarod, while no significant difference was observed between <i>mdx</i> and <i>mdx-Xist</i>^Δhs mice. Average dystrophin level of <i>mdx-Xist</i>^Δhs mice was 21% (2%–45% median 25.8).</p

Serum and plasma biomarkers assessed before and directly after treadmill running.

<p>A. CK levels assessed before and directly after treadmill running. CK levels collected before exercise were significantly (<i>P</i><0.01) lower than those collected directly after exercise in <i>mdx-Xist</i>^Δhs and <i>mdx</i> mice. This exercise induced increase was absent in wild type mice. B. CK levels determined before exercise were significantly (<i>P</i><0.01) elevated in <i>mdx</i> and <i>mdx-Xist</i>^Δhs mice compared to wild type, but this was less pronounced for the <i>mdx-Xist</i>^Δhs mice as <i>mdx</i> mice had significantly (<i>P</i><0.05) higher CK levels. C. Plasma collected directly after exercise contained extremely high CK levels, in both <i>mdx</i> and <i>mdx-Xist</i>^Δhs mice but not in wild type mice. D. Serum levels of MMP-9 were elevated in <i>mdx</i> mice compared to <i>Xist</i>^Δhs mice at both 8 and 14 weeks of age, while levels were normalized in <i>mdx-Xist</i>^Δhs mice. E. TIMP-1 levels were elevated in serum of both <i>mdx</i> and <i>mdx-Xist</i>^Δhs mice. In wild type mice we found an age related increase of the serum TIMP-1 level. Interestingly, at the age of 8 weeks, levels of <i>mdx-Xist</i>^Δhs mice were significantly lower than those of <i>mdx</i> mice. F. <i>Mdx</i> mice had significantly elevated OPN levels compared to both wild type and <i>mdx-Xist</i>^Δhs at 8 weeks of age. Single asterisks indicate a <i>P</i><0.05, average dystrophin level of <i>mdx-Xist</i>^Δhs mice was 21% (2%–45% median 25.8).</p

Muscle fiber size, degree of central nucleation and percentage of fibrotic/necrotic tissue.

<p>A. Regenerating and hypertrophic fibers were mainly observed in the <i>mdx</i> mice. A dystrophin level depend trend towards wild type distribution was observed for <i>mdx-Xist</i>^Δhs mice where <15% dystrophin already resulted in improvement. B. Dystrophin levels between 15–30% and >30% resulted in a reduction of the percentage of centralized nuclei of 40% and 60% respectively. C. The quadriceps of all mice was significantly more severely affected compared to <i>Xist</i>^Δhs mice. D. The diaphragm was the most severely affected muscle with on average 20% fibrotic/necrotic tissue in <i>mdx</i> mice. All mice were significantly more severely affected compared to <i>Xist</i>^Δhs mice. <i>Mdx-Xist</i>^Δhs mice with >30% dystrophin had less fibrotic/necrotic tissue than <i>mdx</i> and <i>mdx-Xist</i>^Δhs mice with <15% dystrophin, but this difference was only significant between both <i>mdx-Xist</i>^Δhs groups. # indicates a significant difference of that bar with all the other groups. Single asterisks indicate a <i>P</i><0.05 and double asterisks indicate a <i>P</i><0.01.</p