The link between mutations in collagen genes and the development of Alport Syndrome has been clearly established and a number of animal models, including knock-out mouse lines, have been developed ...that mirror disease observed in patients. However, it is clear from both patients and animal models that the progression of disease can vary greatly and can be modified genetically. We have identified a point mutation in Col4a4 in mice where disease is modified by strain background, providing further evidence of the genetic modification of disease symptoms. Our results indicate that C57BL/6J is a protective background and postpones end stage renal failure from 7 weeks, as seen on a C3H background, to several months. We have identified early differences in disease progression, including expression of podocyte-specific genes and podocyte morphology. In C57BL/6J mice podocyte effacement is delayed, prolonging normal renal function. The slower disease progression has allowed us to begin dissecting the pathogenesis of murine Alport Syndrome in detail. We find that there is evidence of differential gene expression during disease on the two genetic backgrounds, and that disease diverges by 4 weeks of age. We also show that an inflammatory response with increasing MCP-1 and KIM-1 levels precedes loss of renal function.
Abstract
Aims
Mitochondrial Complex I assembly (MCIA) is a multi-step process that necessitates the involvement of a variety of assembly factors and chaperones to ensure that the final active enzyme ...is correctly assembled. The role of the assembly factor evolutionarily conserved signalling intermediate in the toll (ECSIT) pathway was studied across various murine tissues to determine its role in this process and how this varied between tissues of varying energetic demands. We hypothesized that many of the known functions of ECSIT were unhindered by the introduction of an ENU-induced mutation, while its role in Complex I assembly was affected on a tissue-specific basis.
Methods and results
Here, we describe a mutation in the MCIA factor ECSIT that reveals tissue-specific requirements for ECSIT in Complex I assembly. MCIA is a multi-step process dependent on assembly factors that organize and arrange the individual subunits, allowing for their incorporation into the complete enzyme complex. We have identified an ENU-induced mutation in ECSIT (N209I) that exhibits a profound effect on Complex I component expression and assembly in heart tissue, resulting in hypertrophic cardiomyopathy in the absence of other phenotypes. The dysfunction of Complex I appears to be cardiac specific, leading to a loss of mitochondrial output as measured by Seahorse extracellular flux and various biochemical assays in heart tissue, while mitochondria from other tissues were unaffected.
Conclusions
These data suggest that the mechanisms underlying Complex I assembly and activity may have tissue-specific elements tailored to the specific demands of cells and tissues. Our data suggest that tissues with high-energy demands, such as the heart, may utilize assembly factors in different ways to low-energy tissues in order to improve mitochondrial output. These data have implications for the diagnosis and treatment of various disorders of mitochondrial function as well as cardiac hypertrophy with no identifiable underlying genetic cause.
Graphical Abstract
Graphical Abstract
Collagen I is a member of the Collagen superfamily of proteins, the proteins most abundant in mammals, and an essential component of bones, teeth, skin and connective tissues including ligaments and ...tendons. COL1A1 and COL1A2 are the genes that code for the collagen I alpha chains, α1 and α2 respectively. Collagen I is a heterotrimer of these two alpha chains, formed of two α1 and one α2 chains. Diseases resulting from genetic mutations in COL1A1 and COL1A2, include osteogenesis imperfecta (OI) and Ehlers-Danlos syndrome (EDS), however, mutations in these genes have not been implicated in the development of osteoarthritis (OA).At MRC Harwell Institute, large-scale mutagenesis screens, including the Harwell Ageing Screen, have been used to identify novel models of disease and establish links between genes and diseases. The mutagenised mouse lines MP-107 and TM44 were identified in such screens, exhibiting early-onset mild bone abnormalities at the pelvis and elbow. These animals subsequently developed late-onset phenotypes including abnormal bone growth at the knee and OA.Genetic mapping and sequencing revealed that MP-107 and TM44, contained mutations in Col1a2 and Col1a1 respectively, which correspond to the genes COL1A2 and COL1A1 in humans. The MP-107 mutation was a T to A transversion at position 4521226 of Chromosome 6 resulting in alternative splicing at exon 22 of Col1a2. The TM44 mutation was a C to T transition at position 94836670 of Chromosome 11 resulting in a premature stop codon in exon 31 of Col1a1Extensive phenotyping analysis revealed that the bone abnormalities observed in these lines are a result of an OI phenotype. Evidence of an EDS phenotype was also identified in the line MP-107, indicating that MP-107 is likely a model of Col-1 related overlap syndrome, a proposed EDS subtype exhibiting aspects of OI and EDS. Collagen I related changes to the joint tissues is likely the cause of the OA phenotypes. The project has the potential to enhance understanding of both the development of Col-1 related overlap syndrome and OA, and possible targets for therapy.
Nephrotic syndrome is characterized by severe proteinuria, hypoalbuminaemia, edema and hyperlipidaemia. Genetic studies of nephrotic syndrome have led to the identification of proteins playing a ...crucial role in slit diaphragm signaling, regulation of actin cytoskeleton dynamics and cell-matrix interactions. The laminin α5 chain is essential for embryonic development and, in association with laminin β2 and laminin γ1, is a major component of the glomerular basement membrane, a critical component of the glomerular filtration barrier. Mutations in LAMA5 were recently identified in children with nephrotic syndrome. Here, we have identified a novel missense mutation (E884G) in the uncharacterized L4a domain of LAMA5 where homozygous mice develop nephrotic syndrome with severe proteinuria with histological and ultrastructural changes in the glomerulus mimicking the progression seen in most patients. The levels of LAMA5 are reduced in vivo and the assembly of the laminin 521 heterotrimer significantly reduced in vitro. Proteomic analysis of the glomerular extracellular fraction revealed changes in the matrix composition. Importantly, the genetic background of the mice had a significant effect on aspects of disease progression from proteinuria to changes in podocyte morphology. Thus, our novel model will provide insights into pathologic mechanisms of nephrotic syndrome and pathways that influence the response to a dysfunctional glomerular basement membrane that may be important in a range of kidney diseases.
Display omitted
Abstract
Aims
Cardiac arrhythmias comprise a major health and economic burden and are associated with significant morbidity and mortality, including cardiac failure, stroke, and sudden cardiac death ...(SCD). Development of efficient preventive and therapeutic strategies is hampered by incomplete knowledge of disease mechanisms and pathways. Our aim is to identify novel mechanisms underlying cardiac arrhythmia and SCD using an unbiased approach.
Methods and results
We employed a phenotype-driven N-ethyl-N-nitrosourea mutagenesis screen and identified a mouse line with a high incidence of sudden death at young age (6–9 weeks) in the absence of prior symptoms. Affected mice were found to be homozygous for the nonsense mutation Bcat2p.Q300*/p.Q300* in the Bcat2 gene encoding branched chain amino acid transaminase 2. At the age of 4–5 weeks, Bcat2p.Q300*/p.Q300* mice displayed drastic increase of plasma levels of branch chain amino acids (BCAAs—leucine, isoleucine, valine) due to the incomplete catabolism of BCAAs, in addition to inducible arrhythmias ex vivo as well as cardiac conduction and repolarization disturbances. In line with these findings, plasma BCAA levels were positively correlated to electrocardiogram indices of conduction and repolarization in the German community-based KORA F4 Study. Isolated cardiomyocytes from Bcat2p.Q300*/p.Q300* mice revealed action potential (AP) prolongation, pro-arrhythmic events (early and late afterdepolarizations, triggered APs), and dysregulated calcium homeostasis. Incubation of human pluripotent stem cell-derived cardiomyocytes with elevated concentration of BCAAs induced similar calcium dysregulation and pro-arrhythmic events which were prevented by rapamycin, demonstrating the crucial involvement of mTOR pathway activation.
Conclusions
Our findings identify for the first time a causative link between elevated BCAAs and arrhythmia, which has implications for arrhythmogenesis in conditions associated with BCAA metabolism dysregulation such as diabetes, metabolic syndrome, and heart failure.
Graphical Abstract
Graphical Abstract
We compared commonly used BAPTA-derived chemical Ca2+ dyes (fura2, Fluo-4, and Rhod-2) with a newer genetically encoded indicator (R-GECO) in single cell models of the heart. We assessed their ...performance and effects on cardiomyocyte contractility, determining fluorescent signal-to-noise ratios and sarcomere shortening in primary ventricular myocytes from adult mouse and guinea pig, and in human iPSC-derived cardiomyocytes. Chemical Ca2+ dyes displayed dose-dependent contractile impairment in all cell types, and we observed a negative correlation between contraction and fluorescence signal-to-noise ratio, particularly for fura2 and Fluo-4. R-GECO had no effect on sarcomere shortening. BAPTA-based dyes, but not R-GECO, inhibited in vitro acto-myosin ATPase activity. The presence of fura2 accentuated or diminished changes in contractility and Ca2+ handling caused by small molecule modulators of contractility and intracellular ionic homeostasis (mavacamten, levosimendan, and flecainide), but this was not observed when using R-GECO in adult guinea pig left ventricular cardiomyocytes. Ca2+ handling studies are necessary for cardiotoxicity assessments of small molecules intended for clinical use. Caution should be exercised when interpreting small molecule studies assessing contractile effects and Ca2+ transients derived from BAPTA-like chemical Ca2+ dyes in cellular assays, a common platform for cardiac toxicology testing and mechanistic investigation of cardiac disease physiology and treatment.
Display omitted
•Chemical Ca2+ dyes inhibit contractility in all single cardiomyocytes and reduce force production in loaded cardiac fibres.•Ca2+ dyes inhibit the acto-myosin ATPase with an IC50 ∼5μM, competing with the myosin inhibitor blebbistatin (IC50 ∼1 μM).•Ca2+ transient visibility in single cells is challenging even at dye ∼20 μM.•R-GECO doesn't impair cellular contractility or acto-myosin ATPase activity, generating clear Ca2+ transients at ∼1 μM.•In small molecule assays compound effects of dye plus drug are apparent and mask the drug effect.
Determining the genetic bases of age-related disease remains a major challenge requiring a spectrum of approaches from human and clinical genetics to the utilization of model organism studies. Here ...we report a large-scale genetic screen in mice employing a phenotype-driven discovery platform to identify mutations resulting in age-related disease, both late-onset and progressive. We have utilized N-ethyl-N-nitrosourea mutagenesis to generate pedigrees of mutagenized mice that were subject to recurrent screens for mutant phenotypes as the mice aged. In total, we identify 105 distinct mutant lines from 157 pedigrees analysed, out of which 27 are late-onset phenotypes across a range of physiological systems. Using whole-genome sequencing we uncover the underlying genes for 44 of these mutant phenotypes, including 12 late-onset phenotypes. These genes reveal a number of novel pathways involved with age-related disease. We illustrate our findings by the recovery and characterization of a novel mouse model of age-related hearing loss.
We compared commonly used BAPTA-derived chemical Ca
dyes (fura2, Fluo-4, and Rhod-2) with a newer genetically encoded indicator (R-GECO) in single cell models of the heart. We assessed their ...performance and effects on cardiomyocyte contractility, determining fluorescent signal-to-noise ratios and sarcomere shortening in primary ventricular myocytes from adult mouse and guinea pig, and in human iPSC-derived cardiomyocytes. Chemical Ca
dyes displayed dose-dependent contractile impairment in all cell types, and we observed a negative correlation between contraction and fluorescence signal-to-noise ratio, particularly for fura2 and Fluo-4. R-GECO had no effect on sarcomere shortening. BAPTA-based dyes, but not R-GECO, inhibited in vitro acto-myosin ATPase activity. The presence of fura2 accentuated or diminished changes in contractility and Ca
handling caused by small molecule modulators of contractility and intracellular ionic homeostasis (mavacamten, levosimendan, and flecainide), but this was not observed when using R-GECO in adult guinea pig left ventricular cardiomyocytes. Ca
handling studies are necessary for cardiotoxicity assessments of small molecules intended for clinical use. Caution should be exercised when interpreting small molecule studies assessing contractile effects and Ca
transients derived from BAPTA-like chemical Ca
dyes in cellular assays, a common platform for cardiac toxicology testing and mechanistic investigation of cardiac disease physiology and treatment.
PDF
