The Ras Antagonist, Farnesylthiosalicylic Acid (FTS), Decreases Fibrosis and Improves Muscle Strength in dy2J/dy2J Mouse Model of Muscular Dystrophy

The Ras superfamily of guanosine-triphosphate (GTP)-binding proteins regulates a diverse spectrum of intracellular processes involved in inflammation and fibrosis. Farnesythiosalicylic acid (FTS) is a unique and potent Ras inhibitor which decreased inflammation and fibrosis in experimentally induced liver cirrhosis and ameliorated inflammatory processes in systemic lupus erythematosus, neuritis and nephritis animal models. FTS effect on Ras expression and activity, muscle strength and fibrosis was evaluated in the dy2J/dy2J mouse model of merosin deficient congenital muscular dystrophy. The dy2J/dy2J mice had significantly increased RAS expression and activity compared with the wild type mice. FTS treatment significantly decreased RAS expression and activity. In addition, phosphorylation of ERK, a Ras downstream protein, was significantly decreased following FTS treatment in the dy2J/dy2J mice. Clinically, FTS treated mice showed significant improvement in hind limb muscle strength measured by electronic grip strength meter. Significant reduction of fibrosis was demonstrated in the treated group by quantitative Sirius Red staining and lower muscle collagen content. FTS effect was associated with significantly inhibition of both MMP-2 and MMP-9 activities. We conclude that active RAS inhibition by FTS was associated with attenuated fibrosis and improved muscle strength in the dy2J/dy2J mouse model of congenital muscular dystrophy

Calmodulin Methyltransferase Is Required for Growth, Muscle Strength, Somatosensory Development and Brain Function

<div><p>Calmodulin lysine methyl transferase (CaM KMT) is ubiquitously expressed and highly conserved from plants to vertebrates. CaM is frequently trimethylated at Lys-115, however, the role of CaM methylation in vertebrates has not been studied. <i>CaM KMT</i> was found to be homozygously deleted in the 2P21 deletion syndrome that includes 4 genes. These patients present with cystinuria, severe intellectual disabilities, hypotonia, mitochondrial disease and facial dysmorphism. Two siblings with deletion of three of the genes included in the 2P21 deletion syndrome presented with cystinuria, hypotonia, a mild/moderate mental retardation and a respiratory chain complex IV deficiency. To be able to attribute the functional significance of the methylation of CaM in the mouse and the contribution of <i>CaM KMT</i> to the clinical presentation of the 2p21deletion patients, we produced a mouse model lacking only <i>CaM KMT</i> with deletion borders as in the human 2p21deletion syndrome. No compensatory activity for CaM methylation was found. Impairment of complexes I and IV, and less significantly III, of the mitochondrial respiratory chain was more pronounced in the brain than in muscle. <i>CaM KMT</i> is essential for normal body growth and somatosensory development, as well as for the proper functioning of the adult mouse brain. Developmental delay was demonstrated for somatosensory function and for complex behavior, which involved both basal motor function and motivation. The mutant mice also had deficits in motor learning, complex coordination and learning of aversive stimuli. The mouse model contributes to the evaluation of the role of methylated CaM. CaM methylation appears to have a role in growth, muscle strength, somatosensory development and brain function. The current study has clinical implications for human patients. Patients presenting slow growth and muscle weakness that could result from a mitochondrial impairment and mental retardation should be considered for sequence analysis of the <i>CaM KMT</i> gene.</p></div

Muscle pathology of CaM KMT-/- mice demonstrated by Gomori-Trichrome staining.

<p>The myopathic feature is shown by the variation in fiber size of the two CaM KMT^-/- samples: KO (middle and lower panels), not observed in the sample of the comparable CaM KMT^+/+ WT (upper panel). The yellow arrows point the ragged red fibers in the X40 magnification.</p

Enzymatic activities of respiratory chain complexes.

<p>The enzymatic activities of respiratory chain complexes I,II,II+III and IV were measured by spectrophotometric methods in mitochondria isolated from CaM KMT^+/+ (WT) and CaM KMT^-/- (KO) brain and skeletal (hind limb) muscle. Results are presented as ratio (U/U) to citrate synthase (CS). Each point represents an experiment duplicate performed in different occasions. Significant difference between WT and KO for each is presented above graph (2 tails-student T-Test). For complexes II+III in brain and muscle p values were indicative (0.056 and 0.097, respectively).</p

Construct design and validation of the Knock out mice.

<p>A. Knock out DNA constructs design. The 138 bp starting with the first ATG of the coding sequence of the gene up to the end of the deletion in patients, 300 bp into the adjacent 1^st intron were replaced by a β-gal- Neo cassette of 5600 bp. B. PCR genotyping of the mice. The primers amplifying each of the DNAs are exclusive for that DNA. CaM KMT^-/-: KO, CaM KMT^+/+: WT, CaM KMT^+/-: HET, 1Kb plus DNA ladder (Thermo Scientific): Ma. C. Validation of transcription. RT-PCR on kidney RNA using primers in the 1^st and 2^nd exons of CaM KMT. CaM KMT^-/-: KO, CaM KMT^+/+: WT CaM KMT^+/-: HET, negative control no DNA: n.c, 1kb plus DNA ladder (ThermoSCIENTIFIC):Ma. D. Validation of translation. Liver and kidney homogenates of CaM KMT^-/-:-/-, CaM KMT^+/+: +/+ were analyzed by Western blotting with purified CaM KMT polyclonal antibody [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005388#pgen.1005388.ref011" target="_blank">11</a>]. Adjacent lanes on the same gel contained the indicated amounts of lysates of HEK293 transfected (+) or not transfected (-) with myc-CaM KMT pCDNA3 vector to serve as a marker for the size of CaM KMT. Page ruler prestained protein ladder (#SM1811 Fermentas): Ma. 60 μg of human lymphoblasts and mouse tissues and the indicated amounts of HEK293 lysates were separated on 12% PAA gel. The size of the CaM KMT is indicated by the blue box. Although the polyclonal antibodies were affinity purified, the figure represents many cross reactive proteins. The whole Western blot is presented as supplementary <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005388#pgen.1005388.s003" target="_blank">S3 Fig</a>.</p

Expression of CaM KMT in the brain of adult C57Bl mice.

<p>Representative staining for β-Gal activity sagittal (B) and coronal (D and E) sections of CaM KMT-/- mice brains, compared to CaM KMT+/+ (A and C). The figure represents results of 4 different adult male C57Bl6/J mice brains. Magnified images of cerebellum lobules (F), hippocampus (G), striatum (H) and M1 region of the cerebral cortex (I) Images exemplify the expression in all cells in all the brain regions. The nuclear staining is attributed to the nuclear localization signal in the β galoctosidase gene.</p

Morphogenic development and sensory–motor reflex development of newborn mice.

<p>Developmental milestones were analysed in CaM KMT^+/+ (WT), CaM KMT^-/+ (HET) and CaM KMT^-/- (KO) mice. Newborn weight of male (A) and of female (B). Development of the ability to cling to a vertical wire is presented as the time held on the wire by male and female (C and D, respectively). Newborn mice responsiveness to sensory attraction (E and F). Nest finding score (G and H). Sensory attraction and nest finding scores are represented on a scale from 0 (no response) to 1 (full response in 3 trials). Mean ± SD, n = 7–15 mice in each group. Significant difference between WT and KO is presented above graph (ANOVA post-hoc). For the gaining of weight in parts A and B most of the differences had a p value of 0.000, the less significant one was 0.029. These could not be presented above the graph. Values presented with * were between the KO and HET, due to a small number of the WT that didn't give significance. N = 15–32 (the exact number of mice in each group and test is shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005388#pgen.1005388.s007" target="_blank">S1 Table</a>).</p

Comparison of clinical features of different homozygous deletion in 2P21.

<p>Comparison of clinical features of different homozygous deletion in 2P21.</p

Adult mouse behavior.

<p>Male mice tested were older than 3 months. A. Mice weight. B. The duration of time the mice held to horizontal metal wire grid is shown for each individual mouse. The significant p value is presented (ANOVA post-hoc). The balanced beam was tested in 3 consecutive trials: The time on the beam (C) and the time to reach escape box (D). Mice performance in the rota-rod test in 3 trials: The time the mice stayed on the rotarod (E) and the maximal speed in which the mice successfully stay on the rotarod (F). The significant p values are presented (ANOVA post-hoc). The time mice spent on vibrating platform is shown for WT (G) and KO (H), maximal time on platform was 120 sec. Mean ± SD are presented for C-F, For the balance beam and Rotarod tests: twelve mice for each genotype. CaM KMT^+/+: WT, CaM KMT^+/-: HET, CaM KMT^-/-: KO.</p

Control of tissue expression of CaM KMT and accumulation of hypomethylated CaM in CaM KMT^-/- mice.

<p>A. Control of tissue expression. Activity of β-gal of the targeting cassette under the regulation of endogenous regulation was detected by ONPG analysis on homogenates of the detailed tissues. The results from 3 CaM KMT^-/- and 2 CaM KMT^+/- 3 months old heterozygous male SVJ129 mice are presented. C57Bl6/J mice presented the same pattern. CaM KMT^+/- (HET) and CaM KMT^-/- (KO) B. Accumulation of hypomethylated CaM. Phosphoimage of lystaes of tissues and organs excised from CaM KMT^-/- (KO) or CaM KMT^+/+ (WT) adult females were incubated with <i>Hs</i>CaMKMT and H³ Adomet. The control was recombinant CaM incubated with CaM KMT (C). The figure represents one of three experiments with different female mice of both SVJ129 and C57Bl6/J mice. The numbers at the left side of the figures represent the protein size marker.</p