Circadian rhythm in QT interval is preserved in mice deficient of potassium channel interacting protein 2

Potassium Channel Interacting Protein 2 (KChIP2) is suggested to be responsible for the circadian rhythm in repolarization duration, ventricular arrhythmias, and sudden cardiac death. We investigated the hypothesis that there is no circadian rhythm in QT interval in the absence of KChIP2. Implanted telemetric devices recorded electrocardiogram continuously for 5 days in conscious wild-type mice (WT, n = 9) and KChIP2^?/? mice (n = 9) in light:dark periods and in complete darkness. QT intervals were determined from all RR intervals and corrected for heart rate (QT₁₀₀ = QT/(RR/100)^1/2). Moreover, QT intervals were determined from complexes within the RR range of mean-RR ± 1% in the individual mouse (QT_mean-RR). We find that RR intervals are 125 ± 5 ms in WT and 123 ± 4 ms in KChIP2^?/? (p = 0.81), and QT intervals are 52 ± 1 and 52 ± 1 ms, respectively(p = 0.89). No ventricular arrhythmias or sudden cardiac deaths were observed. We find similar diurnal (light:dark) and circadian (darkness) rhythms of RR intervals in WT and KChIP2^?/? mice. Circadian rhythms in QT₁₀₀ intervals are present in both groups, but at physiological small amplitudes: 1.6 ± 0.2 and 1.0 ± 0.3 ms in WT and KChIP2^?/?, respectively (p = 0.15). A diurnal rhythm in QT₁₀₀ intervals was only found in WT mice. QT_mean-RR intervals display clear diurnal and circadian rhythms in both WT and KChIP2^?/?. The amplitude of the circadian rhythm in QT_mean-RR is 4.0 ± 0.3 and 3.1 ± 0.5 ms in WT and KChIP2^?/?, respectively (p = 0.16). In conclusion, KChIP2 expression does not appear to underlie the circadian rhythm in repolarization duration.     

Circadian photoreception in humans and mice

Circadian rhythms are the endogenous oscillations, occurring with a periodicity of approximately twenty-four hours, in the biochemical and behavioral functions of organisms. In mammals, the phase and period of the rhythm are synchronized to the daily light-dark cycle by light input through the eye. Certain retinal degenerative diseases affecting the photoreceptor cells, both rods and cones, in the outer retina reveal that classical opsins (i.e., rhodopsin and color opsins located in these cells) are essential for vision, but are not required for circadian photoreception. The mammalian cryptochromes and melanopsin (and possibly other opsin family pigments) have been proposed as circadian photoreceptor pigments that exist in the inner retina. Genetic analysis indicates that the cryptochromes, which contain flavin and folate as the light-absorbing cofactors, are the primary circadian photoreceptors. The classical photoreceptors in the outer retina, and melanopsin or other minor opsins in the inner retina, may perform redundant functions in circadian rhythmicity.     

Diazepam improves aspects of social behaviour and neuron activation in NMDA receptor‐deficient mice

NR1 knockdown (NR1KD) mice are genetically modified to express low levels of the NR1 subunit of N‐methyl‐d ‐aspartate (NMDA) receptors, and show deficits in affiliative social behaviour. In this study, we determined which brain regions were selectively activated in response to social stimulation and asked whether differences in neuronal activation could be observed in mice with reduced sociability. Furthermore, we aimed to determine whether brain activation patterns correlated with the amelioration of social deficits through pharmacological intervention. The cingulate cortex, lateral septal nuclei, hypothalamus, thalamus and amygdala showed an increase in c‐Fos immunoreactivity that was selective for exposure to social stimuli. NR1KD mice displayed a reduction in social behaviour and a reduction in c‐Fos immunoreactivity in the cingulate cortex and septal nuclei. Acute clozapine did not significantly alter sociability; however, diazepam treatment did increase sociability and neuronal activation in the lateral septal region. This study has identified the lateral septal region as a neural substrate of social behaviour and the GABA system as a potential therapeutic target for social dysfunction.     

Circadian rhythm of irinotecan tolerability in mice

The toxicity of irinotecan (CPT-11), a topoisomerase-I inhibitor largely used in cancer patients, was investigated as a function of the circadian time of its administration in mice, with mortality, body weight loss, leukopenia, neutropenia, intestinal lesions, and bone marrow cell cycle phase distribution as end points. Four experiments were performed on a total of 773 male mice standardized with 12h light/12h darkness. Irinotecan was administered daily for 4 or 10 consecutive days (D1-4 and D1-10, respectively, in different experiments) at one of six circadian stages expressed in hours after light onset (HALO). The survival curves differed significantly as a function of the dosage and circadian time of drug administration by the D1-10 schedule, with 70% survival at 7 or 11 HALO and 51% at 19 or 23 HALO (p=0.039 from log rank test). CPT-11 administration at 19 or 23 HALO resulted in (1) greatest mean body weight loss at nadir; (2) most severe colic and bone marrow lesions and/or slowest recovery; and (3) deepest neutropenia nadir and/or slowest hematologic recovery. These circadian treatment time-related differences were statistically validated. The bone marrow cell cycle data revealed a four to eight-fold larger G2-M phase arrest following irinotecan administration at 19 or 23 HALO in comparison to the other times of drug administration, apparently representative of the repair of more extensive DNA damage (p < 0.001 from ANOVA) when the medication was given at these circadian times. Overall, CPT-11 was better tolerated by mice treated during the light (animals' rest) span. The results support the administration of CPT-11 to cancer patients in the second half of the night, during sleep, in order to improve drug tolerability.     

Oxytocin hypersensitivity in pregnant P-LAP deficient mice

AimsOxytocin (OT) is the strongest uterotonic substance and has been used widely to induce labor. The physiological importance of OT in modulating the initiation and progression of labor remains unclear. In this study, we showed the roles of OT with onset of labor and also the arginine vasopressin (AVP) effect on urine volume in vivo using both wild type (WT) and placental leucine aminopeptidase (P-LAP)-deficient (KO) mice.Main methodsOT (1, 2, 2.5 U/day) or recombinant P-LAP (0.01 U/day) was continuously infused from gestation day 15.5 in WT and P-LAP KO mice. Duration until onset of labor was observed. Before and after administration of AVP (1 U/day) in WT and P-LAP KO mice, urine volume was measured.Key findingsA significant shortening of pregnancy term was observed in P-LAP KO mice. Continuous infusion of OT (1 U/day) revealed that P-LAP KO mice resulted in premature delivery (OT hypersensitivity). We could observe a significant decrease of urine volume in P-LAP KO mice by administration of AVP. Administration of recombinant P-LAP in WT mice resulted in the delay of the onset of labor about 1.5 days compared with control mice.SignificanceOur present study shows that the regulation of the onset of labor mainly depends on OT and its degradation by P-LAP and also the possible role of P-LAP in the regulation of urine output. P-LAP might be involved in the increased OT sensitivity just prior to onset of labor and also in the onset of labor by degradation of OT.     

Impaired melanoma growth in VASP deficient mice

Progression of tumors depends on interactions of cancer cells with the host environment. Expression of the cytoskeleton protein VASP is upregulated in various cancer entities. We analyzed the role of VASP for melanoma growth in murine allograft models. Growth of VASP expressing melanomas was retarded in VASP^?/? versus wild-type animals. Over time tumor size was <50% in VASP^?/? versus wild-type animals and independent of expression levels of Ena/VASP protein family members. Histological analyses showed smaller cells with impaired nutrition status and less vascularization in melanomas derived from VASP^?/? versus counterparts from wild-type mice. Cumulatively, the data reveal a critical role of VASP in non-tumor cells in the tumor environment for melanoma growth in vivo.     

Bleomycin-induced pulmonary injury in mice deficient in SPARC

SPARC (secreted protein, acidic and rich in cysteine) is a component of the matrix that appears to regulate tissue remodeling. There is evidence that it accumulates in the lung in the setting of pulmonary injury and fibrosis, but direct evidence of its involvement is only now emerging. We therefore investigated the development of pulmonary fibrosis induced by bleomycin administered either intratracheally or intraperitoneally in mice deficient in SPARC. Bleomycin (0.15 U/mouse) given intratracheally induced significantly more pulmonary fibrosis in mice deficient in SPARC compared with that in wild-type control mice, with the mutant mice demonstrating greater neutrophil accumulation in the lung. However, in wild-type and SPARC-deficient mice given intraperitoneal bleomycin (0.8 U/injection x 5 injections over 14 days), the pattern and severity of pulmonary fibrosis, as well as the levels of leukocyte recruitment, were similar in both strains of mice. These findings suggest that the involvement of SPARC in pulmonary injury is likely to be complex, dependent on several factors including the type, duration, and intensity of the insult. Furthermore, increased neutrophil accumulation in the peritoneal cavity was also observed in SPARC-null mice after acute chemical peritonitis. Together, these data suggest a possible role for SPARC in the recruitment of neutrophils to sites of acute inflammation.     

Neurogenic inflammation in mice deficient in heparin-synthesizing enzyme

Mast cell activation, or neurogenic inflammation, is known to induce lowering of interstitial fluid pressure (P(if)) and plasma protein extravasation (PPE) in several tissues from both rats and mice. To examine a possible role of connective tissue mast cells (CTMCs) in these inflammatory responses, we used mice with dysfunctional CTMCs due to lack of the N-deacetylase/N-sulfotransferase-2 enzyme (NDST-2(-/-)). P(if) and PPE were measured after challenge with compound 48/80 (C48/80), and P(if) alone was measured after treatment either with capsaicin, substance P (SP), or calcitonin gene-related peptide (CGRP). Measurements of P(if) in anesthetized (fentanyl/fluanison and midazolam, 1:1) mice were performed in paw skin with glass capillaries connected to a servo-controlled counterpressure system. PPE was measured with microdialysis by using hollow plasmapheresis fibers (cutoff at 3,000 kDa) placed subcutaneously on the back. Intravenous administration of C48/80 lowered P(if) significantly (P < 0.05) in NDST-2(-/-) mice (-1.67 +/- 0.42 mmHg) compared with vehicle (-0.57 +/- 0.17 mmHg) but the lowering was significantly (P < 0.05) less compared with that of the NDST-2(+/+) mice (-2.31 +/- 0.47 mmHg). PPE was increased 300% after treatment with C48/80 in NDST-2(+/+) mice, whereas there was no increase in PPE in NDST-2(-/-) mice. Capsaicin, SP, and CGRP lowered P(if) significantly (P < 0.05) compared with vehicle and to the same extent in both NDST-2(+/+) and NDST-2(-/-) mice. We can conclude that although NDST-2(-/-) mice demonstrate an altered response in P(if) after mast cell activation, there was no similar alteration after neurogenic inflammation. Therefore, we suggest that neurogenic inflammation in mouse skin is not exclusively dependent on intact CTMCs.     

Dorsal pancreas agenesis in N-cadherin- deficient mice.

Members of the cadherin family of cell adhesion molecules are thought to be crucial regulators of tissue patterning and organogenesis. During pancreatic ontogeny N-cadherin is initially expressed in the pancreatic mesenchyme and later in pancreatic endoderm. Analysis of N-cadherin-deficient mice revealed that these mice suffer from selective agenesis of the dorsal pancreas. Further analysis demonstrated that the mechanism for the lack of a dorsal pancreas involves an essential function of N-cadherin as a survival factor in the dorsal pancreatic mesenchyme.     

Early embryonic death of mice deficient in gamma-adaptin

Intracellular protein transport and sorting by vesicles in the secretory and endocytic pathways requires the formation of a protein coat on the membrane. The heterotetrameric adaptor protein complex 1 (AP-1) promotes the formation of clathrin-coated vesicles at the trans-Golgi network. AP-1 interacts with various sorting signals in the cytoplasmic tails of cargo molecules, thus indicating a function in protein sorting. We generated mutants of the gamma-adaptin subunit of AP-1 in mice to investigate its role in post-Golgi vesicle transport and sorting processes. gamma-Adaptin-deficient embryos develop until day 3.5 post coitus and die during the prenidation period, revealing that AP-1 is essential for viability. In heterozygous mice the amount of AP-1 complexes is reduced to half of controls. Free beta1- or micro1 chains were not detectable, indicating that they are unstable unless they are part of AP-1 complexes. Heterozygous mice weigh less then their wild-type littermates and show impaired T cell development.     

Enhanced vasoconstrictor responses in eNOS deficient mice.

Previous studies suggest that vasoconstriction is modulated by nitric oxide (NO). Contractions to ET-1 and/or thromboxane may be enhanced during chronic deficiency in expression or activity of NO synthase (NOS). Multiple isoforms of NOS are expressed within the vessel wall and purely pharmacological approaches cannot define the role of each. We tested the hypothesis that vasoconstriction to endothelin-1 (ET-1) and/or the thromboxane mimetic, U46619, is enhanced under conditions of chronic, selective deficiency in endothelial NOS (eNOS-/-) by examining responses in aorta from eNOS-/- mice compared to wild type (eNOS+/+). ET-1 produced dose-dependent contraction of aorta from eNOS+/+ mice that was increased twofold following acute inhibition of all NOS isoforms with N(G)-nitro-L-arginine (L-NNA). In eNOS-/- mice, contractions to ET-1 were increased twofold compared to eNOS+/+. L-NNA had no effect. Although contraction of the aorta to thromboxane mimetic U46619 was increased at lower concentrations, maximal contractions to U46619 were not increased following acute inhibition of NOS or in eNOS-/- mice. These studies provide direct evidence that vasoconstriction to ET-1 and thromboxane is augmented in the face of eNOS deficiency, demonstrating that eNOS normally inhibits vascular contractile responses.     

Molar tooth development in caspase-3 deficient mice

Tooth morphogenesis is accompanied by apoptotic events which show restricted temporospatial patterns suggesting multiple roles in odontogenesis. Dental apoptosis seems to be caspase dependent and caspase-3 has been shown to be activated during dental apoptosis.Caspase-3 mutant mice on different genetic backgrounds were used to investigate alterations in dental apoptosis and molar tooth morphogenesis. Mouse embryos at E15.5 were analyzed to reveal any changes in enamel knots, which are transient structures eliminated by apoptosis. In caspase-3(-/-) mice on the B57BL/6 background, disorganization of the epithelium was found in the original primary enamel knot area and confirmed by altered expression of Shh. Despite this early defect in molar tooth development, these mutants showed correct formation of secondary enamel knots as indicated by Fgf-4 expression. Analyses of adult molar teeth did not reveal any major alterations in tooth shape, enamel structure or pattern when compared to heterozygote littermates. In caspase-3(-/-) mice on the 129X1/SvJ background, no defects in tooth development were found except the position of the upper molars which developed more posteriorly in the oral cavity. This is likely, however, to be a secondary defect caused by a physical squashing of the face by the malformed brain. The results suggest that although caspase-3 becomes activated and may be essential for dental apoptosis, it does not seem fundamental for formation of normal mineralised molar teeth.     

Circadian Disruption: comparing humans with mice

Disruption of the 24-h light-dark cycle has been implicated as an endocrine disruptor and linked to increased morbidity and mortality in animal studies. Previously reported measurements of circadian disruption in day-shift and rotating-shift nurses were compared with new mouse data where the light-dark patterns simulated shiftwork. Phasor magnitudes, a measure of circadian entrainment, were shown to be similar for humans and for mice when exposed to similar patterns of light and dark. Phasor analyses may be a useful method for quantitatively bridging ecological measurements of circadian disruption in human with parametric studies of health outcomes in a mouse model.