Mutations in a number of genes have been linked to inherited dilated cardiomyopathy (DCM). However, such mutations account for only a small proportion of the clinical cases emphasising the need for alternative discovery approaches to uncovering novel pathogenic mutations in hitherto unidentified pathways. Accordingly, as part of a large-scale N-ethyl-N-nitrosourea mutagenesis screen, we identified a mouse mutant, Python, which develops DCM. We demonstrate that the Python phenotype is attributable to a dominant fully penetrant mutation in the dynamin-1-like (Dnm1l) gene, which has been shown to be critical for mitochondrial fission. The C452F mutation is in a highly conserved region of the M domain of Dnm1l that alters protein interactions in a yeast two-hybrid system, suggesting that the mutation might alter intramolecular interactions within the Dnm1l monomer. Heterozygous Python fibroblasts exhibit abnormal mitochondria and peroxisomes. Homozygosity for the mutation results in the death of embryos midway though gestation. Heterozygous Python hearts show reduced levels of mitochondria enzyme complexes and suffer from cardiac ATP depletion. The resulting energy deficiency may contribute to cardiomyopathy. This is the first demonstration that a defect in a gene involved in mitochondrial remodelling can result in cardiomyopathy, showing that the function of this gene is needed for the maintenance of normal cellular function in a relatively tissue-specific manner. This disease model attests to the importance of mitochondrial remodelling in the heart; similar defects might underlie human heart muscle disease.
BackgroundFibrillins 1 (FBN1) and 2 (FBN2) are components of microfibrils, microfilaments that are present in many connective tissues, either alone or in association with elastin. Marfan's syndrome and congenital contractural arachnodactyly (CCA) result from dominant mutations in the genes FBN1 and FBN2 respectively. Patients with both conditions often present with specific muscle atrophy or weakness, yet this has not been reported in the mouse models. In the case of Fbn1, this is due to perinatal lethality of the homozygous null mice making measurements of strength difficult. In the case of Fbn2, four different mutant alleles have been described in the mouse and in all cases syndactyly was reported as the defining phenotypic feature of homozygotes.Methodology/Principal FindingsAs part of a large-scale N-ethyl-N-nitrosourea (ENU) mutagenesis screen, we identified a mouse mutant, Mariusz, which exhibited muscle weakness along with hindlimb syndactyly. We identified an amber nonsense mutation in Fbn2 in this mouse mutant. Examination of a previously characterised Fbn2-null mutant, Fbn2fp, identified a similar muscle weakness phenotype. The two Fbn2 mutant alleles complement each other confirming that the weakness is the result of a lack of Fbn2 activity. Skeletal muscle from mutants proved to be abnormal with higher than average numbers of fibres with centrally placed nuclei, an indicator that there are some regenerating muscle fibres. Physiological tests indicated that the mutant muscle produces significantly less maximal force, possibly as a result of the muscles being relatively smaller in Mariusz mice.ConclusionsThese findings indicate that Fbn2 is involved in integrity of structures required for strength in limb movement. As human patients with mutations in the fibrillin genes FBN1 and FBN2 often present with muscle weakness and atrophy as a symptom, Fbn2-null mice will be a useful model for examining this aspect of the disease process further.
Inflammatory responses are induced by members of the Toll-like and Interleukin-1 receptor family. We have identified an IL-1RI (interleukin-1 receptor type I) co-receptor, TILRR, (toll-like and IL-1 receptor regulator), which associates with the signalling receptor and potentiates activation of NF-kB and inflammatory responses.1 Earlier studies have confirmed expression and function of TILRR in both inflammatory and vascular cells. More recent experiments have demonstrated a pronounced increase in TILRR expression in the atherosclerotic plaque using mouse models (ApoE−/− and LDLR−/−). The current studies focus on determining the role of TILRR in development of atherosclerosis, specifically in relation to its impact on IL-1-induced responses. Immuno-histochemical studies have demonstrated the presence of TILRR in sections of vascular lesions from human samples, correlating with areas of IL-1RI activity. In experiments using ApoE/IL-1RI double knockouts, the high fat diet had only minor impact on the level of TILRR expression in vascular tissue. We have confirmed specificity of the co-receptor to IL-RI by demonstrating that other receptor systems controlling vascular inflammation, such as TNFR, are insensitive to TILRR expression over a range of ligand concentrations. Ongoing studies use custom anti-TILRR antibodies to test effects of blocking TILRR association with IL-1RI on development of atherosclerosis. Antibodies are injected into fat-fed ApoE−/− mice, and plaque formation determined using computer based imaging and semi-quantitative scoring.
Method Male Wistar rats at 6 months (young), 12 months (adult) and 24 months (old) (n=5 per group) were killed by a Home Office approved. The SAN region of the heart was dissected and maintained in bicarbonate-buffered saline at 37°C. Intrinsic pacemaker activity was recorded under control conditions and in the presence of 3µMM cyclopiazonic acid (CPA) to inhibit SERCA. Western blot was used to assess expression of SERCA and the SR calcium-release channel, the ryanodine receptor RYR2, both shown expressed relative to levels in the young SAN. Data are shown as mean ± SEM. Results Intrinsic SAN beating rate significantly decreased in old age (young 260±17bpm vs. old 216±15bpm; P=0.04). CPA caused slowing in both young and adult SAN by 26±9 and 49±10bpm respectively (P=0.001), but not in the old SAN. SERCA2a and RYR2 expression increased from young to adult (SERCA young 100±9.8% vs. adult 135±10.8%; P=0.002: RYR2 young 100±21.9% vs. 152±11.2%; p=0.0006), but declined substantially in old age to levels below the young (SERCA old 72±11.7%; RYR2 old 53±4.3%). Conclusion The data show a diminished SR influence on pacemaking in the old SAN. In contrast developmentally the SR may increase its pacemaker role from young to adult animals. Toll-like and IL-1 receptors control inflammatory responses. TILRR (Toll-like IL-1 Receptor Regulator), is an IL-1 co-receptor which associates with the type I IL-1 receptor (IL-1RI) to amplify activation of NF-kappaB and inflammatory responses. Earlier studies have demonstrated increased levels of TILRR in the atherosclerotic plaque. Further, that injection of a polyclonal anti-TILRR antibody, which blocks TILRR/IL-1RI association and reduces inflammatory responses, causes a 25% decrease in plaque formation in ApoE-/-mice on a high fat diet. Alanine scanning mutagenesis identified two sites within the TILRR core protein, which allow distinct control of Il-1 activities. The R425A substitution blocks enhanced cell survival, but functions as wild-type in relation to inflammatory responses. In contrast, a D448A substitution reduces MyD88-dependent inflammatory responses, but has no impact on cell survival [1]. Current studies use peptides designed to block these distinct functional sites to further analyse consequences of selective inhibition on downstream events. Results show a successive reduction in inflammatory responses by the peptide designed to block D448 dependent interactions, with no effect of the peptide targeting anti-apoptotic signals or a non-specific control. Ongoing studies are testing the effect of the peptides on IL-1-induced cell survival. TILRR FUNCTIONAL MUTANTS SELECTIVELY INHIBIT INFLAMMATORY AND ANTI-APOPTOTIC RESPONSES
Inflammatory responses are induced by members of the Toll-Like and interleukin (IL)-1 receptor family and controlled by NF-κB.We have identified an IL-1RI co-receptor, TILRR, which potentiates activation of NF-κB and inflammatory responses. We show that induction of amplification depends on formation of a TILRR-containing receptor complex, which imparts enhanced recruitment of the MyD88 adaptor to the signalling receptor IL-1RI, and induction of Ras-dependent amplification of NF-κB.1We have confirmed expression of TILRR in vascular cells and have demonstrated a correlation of the level of TILRR expression with the level of NF-κB activity and inflammatory responses, induced by IL-1 stimulation and by mechanotransduction.Our recent studies have demonstrated expression of TILRR in vascular endothelial cells using immunohistochemistry. Sections of perfusion-fixed, paraffin-embedded vascular tissue were stained using a specific rabbit polyclonal anti-TILRR antibody, followed by incubation with a biotinylated goat anti-rabbit antibody. Ongoing studies using wild type and ApoE−/− mice are designed to assess the impact of diet on TILRR expression and on its association with the signalling partner IL-1RI.
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