Mice expressing a human K(ATP) channel mutation have altered channel ATP sensitivity but no cardiac abnormalities.

AIMS/HYPOTHESIS: Patients with severe gain-of-function mutations in the Kir6.2 subunit of the ATP-sensitive potassium (K(ATP)) channel, have neonatal diabetes, muscle hypotonia and mental and motor developmental delay-a condition known as iDEND syndrome. However, despite the fact that Kir6.2 forms the pore of the cardiac K(ATP) channel, patients show no obvious cardiac symptoms. The aim of this project was to use a mouse model of iDEND syndrome to determine whether iDEND mutations affect cardiac function and cardiac K(ATP) channel ATP sensitivity. METHODS: We performed patch-clamp and in vivo cine-MRI studies on mice in which the most common iDEND mutation (Kir6.2-V59M) was targeted to cardiac muscle using Cre-lox technology (m-V59M mice). RESULTS: Patch-clamp studies of isolated cardiac myocytes revealed a markedly reduced K(ATP) channel sensitivity to MgATP inhibition in m-V59M mice (IC(50) 62 µmol/l compared with 13 µmol/l for littermate controls). In vivo cine-MRI revealed there were no gross morphological differences and no differences in heart rate, end diastolic volume, end systolic volume, stroke volume, ejection fraction, cardiac output or wall thickening between m-V59M and control hearts, either under resting conditions or under dobutamine stress. CONCLUSIONS/INTERPRETATION: The common iDEND mutation Kir6.2-V59M decreases ATP block of cardiac K(ATP) channels but was without obvious effect on heart function, suggesting that metabolic changes fail to open the mutated channel to an extent that affects function (at least in the absence of ischaemia). This may have implications for the choice of sulfonylurea used to treat neonatal diabetes.

Igf2 ligand dependency of Pten(+/-) developmental and tumour phenotypes in the mouse.

The tumour suppressor PTEN is a key negative regulator of the PI3K-Akt pathway, and is frequently either reduced or lost in human tumours. Murine genetic studies have confirmed that reduction of Pten promotes tumourigenesis in multiple organs, and demonstrated dependency of tumour development on the activation of downstream components such as Akt. Insulin-like growth factors (IGFs) act via IGF1R to activate the PI3K-Akt pathway, and are commonly upregulated in cancer. A context-dependent interplay between IGFs and PTEN exists in normal tissue and tumours; increased IGF2 ligand supply induces Pten expression creating an autoregulatory negative feedback loop, whereas complete loss of PTEN may either cooperate with IGF overexpression in tumour promotion, or result in desensitisation to IGF ligand. However, it remains unknown whether neoplasia associated with Pten loss is dependent on upstream IGF ligand supply in vivo. We evaluated this by generation of Pten(+/-) mice with differing allelic dosage of Igf2, an imprinted gene encoding the potent embryonic and tumour growth factor Igf2. We show that biallelic Igf2 supply potentiates a previously unreported Pten(+/-) placental phenotype and results in strain-dependent cardiac hyperplasia and neonatal lethality. Importantly, we also show that the effects of Pten loss in vivo are modified by Igf2 supply, as lack of Igf2 results in extended survival and delayed tumour development while biallelic supply is associated with reduced lifespan and accelerated neoplasia in females. Furthermore, we demonstrate that reduction of PTEN protein to heterozygote levels in human MCF7 cells is associated with increased proliferation in response to IGF2, and does not result in desensitisation to IGF2 signalling. These data indicate that the effects of Pten loss at heterozygote levels commonly observed in human tumours are modified by Igf2 ligand, and emphasise the importance of the evaluation of upstream pathways in tumours with Pten loss.

Measurement and analysis of sarcomere length in rat cardiomyocytes in situ and in vitro.

Sarcomere length (SL) is an important determinant and indicator of cardiac mechanical function; however, techniques for measuring SL in living, intact tissue are limited. Here, we present a technique that uses two-photon microscopy to directly image striations of living cells in cardioplegic conditions, both in situ (Langendorff-perfused rat hearts and ventricular tissue slices, stained with the fluorescent marker di-4-ANEPPS) and in vitro (acutely isolated rat ventricular myocytes). Software was developed to extract SL from two-photon fluorescence image sets while accounting for measurement errors associated with motion artifact in raster-scanned images and uncertainty of the cell angle relative to the imaging plane. Monte-Carlo simulations were used to guide analysis of SL measurements by determining error bounds as a function of measurement path length. The mode of the distribution of SL measurements in resting Langendorff-perfused heart is 1.95 mum (n = 167 measurements from N = 11 hearts) after correction for tissue orientation, which was significantly greater than that in isolated cells (1.71 mum, n = 346, N = 9 isolations) or ventricular slice preparations (1.79 mum, n = 79, N = 3 hearts) under our experimental conditions. Furthermore, we find that edema in arrested Langendorff-perfused heart is associated with a mean SL increase; this occurs as a function of time ex vivo and correlates with tissue volume changes determined by magnetic resonance imaging. Our results highlight that the proposed method can be used to monitor SL in living cells and that different experimental models from the same species may display significantly different SL values under otherwise comparable conditions, which has implications for experiment design, as well as comparison and interpretation of data.

Isoproterenol induces in vivo functional and metabolic abnormalities: similar to those found in the infarcted rat heart.

Chronic isoproterenol administration produces a rapid, highly reproducible rodent model of cardiac hypertrophy. Yet, despite widespread use of this model, the effects of isoproterenol on in vivo cardiac function and substrate metabolism are unknown. Isoproterenol (5 mg.kg(-1).day(-1)) was infused for 7 days in male Wistar rats (n = 22). In vivo magnetic resonance imaging (MRI) showed that left ventricular mass increased by 37% and end-diastolic and systolic volumes increased by 33% and 73%, respectively, following isoproterenol infusion. Cardiac function at the base of the left ventricle was normal, but apical ejection fraction decreased from 90% to 31% and apical free wall thickening decreased by 94%, accompanied by increased fibrosis and inflammation. Myocardial palmitate oxidation rates were 25% lower, and citrate synthase and medium chain acyl-coenzyme A dehydrogenase activities were reduced by 25% and 29%, respectively, following isoproterenol infusion. Fatty acid transporter protein levels were 11-52% lower and triglyceride concentrations were 55% lower in isoproterenol-infused rat hearts. Basal glycolysis and glycogen concentration were not changed, yet insulin stimulated glycolysis was decreased by 32%, accompanied by 33% lower insulin stimulated glucose transporter, GLUT4, protein levels in rat hearts following isoproterenol infusion, compared with controls. In conclusion, isoproterenol infusion impaired in vivo cardiac function, induced hypertrophy, and decreased both fatty acid and glucose metabolism, changes similar in direction and magnitude to those found in the rat heart following moderate severity myocardial infarction.

MRS reveals additional hexose N-acetyl resonances in the brain of a mouse model for Sandhoff disease.

Sandhoff disease, one of several related lysosomal storage disorders, results from the build up of N-acetyl-containing glycosphingolipids in the brain and is caused by mutations in the genes encoding the hexosaminidase beta-subunit. Affected individuals undergo progressive neurodegeneration in response to the glycosphingolipid storage. (1)H magnetic resonance spectra of perchloric acid extracts of Sandhoff mouse brain exhibited several resonances ca 2.07 ppm that were not present in the corresponding spectra from extracts of wild-type mouse brain. High-performance liquid chromatography and mass spectrometry of the Sandhoff extracts post-MRS identified the presence of N-acetylhexosamine-containing oligosaccharides, which are the likely cause of the additional MRS resonances. MRS of intact brain tissue with magic angle spinning also showed additional resonances at ca 2.07 ppm in the Sandhoff case. These resonances appeared to increase with disease progression and probably arise, for the most part, from the stored glycosphingolipids, which are absent in the aqueous extracts. Hence in vivo MRS may be a useful tool for detecting early-stage Sandhoff disease and response to treatment.

Detection of the inhibitory neurotransmitter GABA in macrophages by magnetic resonance spectroscopy.

Macrophages are key components of the inflammatory response to tissue injury, but their activities can exacerbate neuropathology. High-resolution magnetic resonance spectroscopy was used to identify metabolite levels in perchloric acid extracts of cultured cells of the RAW 264.7 murine macrophage line under resting and lipopolysaccharide-activated conditions. Over 25 metabolites were identified including gamma-aminobutyric acid (GABA), an inhibitory neurotransmitter not previously reported to be present in macrophages. The presence of GABA was also demonstrated in extracts of human peripheral blood monocyte-derived macrophages. This finding suggests that there may be communication between damaged central nervous system (CNS) tissue and recruited macrophages and resident microglia, which could help orchestrate the immune response. On activation, lactate, glutamine, glutamate, and taurine levels were elevated significantly, and GABA and alanine were reduced significantly. Strong resonances from glutathione, evident in the macrophage two-dimensional 1H spectrum, suggest that this may have potential as a noninvasive marker of macrophages recruited to the CNS, as it is only present at low levels in normal brain. Alternatively, a specific combination of spectroscopic changes, such as lactate, alanine, glutathione, and polyamines, may prove to be the most accurate means of detecting macrophage recruitment to the CNS.