Myosin expression and contractile function are altered by replating stem cell-derived cardiomyocytes.

Myosin heavy chain (MyHC) is the main determinant of contractile function. Human ventricular cardiomyocytes (CMs) predominantly express the ß-isoform. We previously demonstrated that ∼80% of human embryonic stem cell-derived cardiomyocytes (hESC-CMs) express exclusively ß-MyHC after long-term culture on laminin-coated glass coverslips. Here, we investigated the impact of enzymatically detaching hESC-CMs after long-term culture and subsequently replating them for characterization of cellular function. We observed that force-related kinetic parameters, as measured in a micromechanical setup, resembled α- rather than ß-MyHC-expressing myofibrils, as well as changes in calcium transients. Single-cell immunofluorescence analysis revealed that replating hESC-CMs led to rapid upregulation of α-MyHC, as indicated by increases in exclusively α-MyHC- and in mixed α/ß-MyHC-expressing hESC-CMs. A comparable increase in heterogeneity of MyHC isoform expression was also found among individual human induced pluripotent stem cell (hiPSC)-derived CMs after replating. Changes in MyHC isoform expression and cardiomyocyte function induced by replating were reversible in the course of the second week after replating. Gene enrichment analysis based on RNA-sequencing data revealed changes in the expression profile of mechanosensation/-transduction-related genes and pathways, especially integrin-associated signaling. Accordingly, the integrin downstream mediator focal adhesion kinase (FAK) promoted ß-MyHC expression on a stiff matrix, further validating gene enrichment analysis. To conclude, detachment and replating induced substantial changes in gene expression, MyHC isoform composition, and function of long-term cultivated human stem cell-derived CMs, thus inducing alterations in mechanosensation/-transduction, that need to be considered, particularly for downstream in vitro assays.

Fostered offspring develop hyper-reactive endocrine stress responses in a plural-breeding rodent, Octodon degus.

Long-term parental separation can lead to altered behavioral and physical development in human children. Rodent models are popular for studying parent-child separation, and several studies have found that maternal separation leads to chronic changes in the endocrine stress response. However, while human children are generally raised by multiple caregivers, most rodent studies utilize solitary breeding species. Therefore, we used degus (Octodon degus) as a model for studying human parental separation, as these rodents practice plural breeding and communal care. In this study, we cross-fostered degu litters at different ages (post-natal day [PND] 2, 8, and 14) to test the hypotheses that fostering affects offspring stress hormone levels in both the short- and long-term and that these impacts differ depending on the age at which offspring are fostered. We found that fostering had long-term effects, as fostered offspring had higher stress-induced cortisol levels and weaker cortisol negative feedback than non-fostered offspring at weaning age (PND28). We also found that the timing of fostering mattered, as degus fostered at PND8 had higher baseline cortisol levels the day after fostering, while degus fostered at PND2 had higher stress-induced cortisol levels at weaning. These data suggest that long-term cross-fostering has enduring impacts on the endocrine stress response in degus, therefore making them a useful model organism for investigating impacts of parental separation in humans.

Plan Selection in Proton Therapy of Locally Advanced Prostate Cancer with Simultaneous Treatment of Multiple Targets.

PURPOSE: Intensity modulated proton therapy (IMPT) of locally advanced prostate cancer can spare the bowel considerably compared with modern photon therapy, but simultaneous treatment of the prostate (p), seminal vesicles (sv), and lymph nodes is challenging owing to day-to-day organ motion and range uncertainties. Our purpose was, therefore, to generate a plan library for use in adaptive IMPT to mitigate these uncertainties. METHODS AND MATERIALS: We retrospectively included 27 patients with a series of computed tomography scans throughout their treatment representing day-to-day variation. In 18 of the patients, target motion was analyzed using rigid shifts of prostate gold markers relative to bony anatomy. A plan library with different p and sv planning target volume (p/sv-PTV) positions was defined from the distribution and direction of these shifts. Delivery of IMPT using plan selection from the library was simulated for image guidance on bony anatomy, in the remaining patients and compared with nonadaptive IMPT. RESULTS: The plan library consisted of 3 small margin p/sv-PTVs: (1) p/sv-PTV shifted 1.5 systematic error (Σ) of the population mean in the anterior and cranial directions, (2) p/sv-PTV shifted 1.5Σ in the posterior and caudal directions, and (3) p/sv-PTV in the planning position. The conventional p/sv-PTV was also available for backup. Plan selection compared with nonadaptive IMPT resulted in a reduction of the rectum volume receiving 60 Gy relative biological effect (RBE) (V60GyRBE) from on average 12 mL to 9 mL. For the bladder the average V45GyRBE was reduced from 36% to 30%. Large and small bowel doses were also reduced, whereas target coverage was comparable or improved compared with nonadaptive IMPT. CONCLUSIONS: Plan selection based on a population model of rigid target motion was feasible for all patients. Compared with conventional IMPT, plan selection resulted in significant dosimetric sparing of rectum and bladder without compromising target coverage.

Differential cerebellar GABAA receptor expression in mice with mutations in CaV2.1 (P/Q-type) calcium channels.

Ataxia is the predominant clinical manifestation of cerebellar dysfunction. Mutations in the human CACNA1A gene, encoding the pore-forming α1 subunit of CaV2.1 (P/Q-type) calcium channels, underlie several neurological disorders, including Episodic Ataxia type 2 and Familial Hemiplegic Migraine type 1 (FHM1). Several mouse mutants exist that harbor mutations in the orthologous Cacna1a gene. The spontaneous Cacna1a mutants Rolling Nagoya (tg(rol)), Tottering (tg) and Leaner (tg(ln)) mice exhibit behavioral motor phenotypes, including ataxia. Transgenic knock-in (KI) mouse strains with the human FHM1 R192Q and S218L missense mutations have been generated. R192Q KI mice are non-ataxic, whereas S218L KI mice display a complex behavioral phenotype that includes cerebellar ataxia. Given the dependence of γ-aminobutyric acid type A (GABAA) receptor subunit functioning on localized calcium currents, and the functional link between GABAergic inhibition and ataxia, we hypothesized that cerebellar GABAA receptor expression is differentially affected in Cacna1a mutants and contributes to the ataxic phenotype. Herein we quantified functional GABAA receptors and pharmacologically dissociated cerebellar GABAA receptors in several Cacna1a mutants. We did not identify differences in the expression of GABAA receptor subunits or in the number of functional GABAA receptors in the non-ataxic R192Q KI strain. In contrast, tg(rol) mice had a ∼15% decrease in the number of functional GABAA receptors, whereas S218L KI mice showed a ∼29% increase. Our data suggest that differential changes in cerebellar GABAA receptor expression profile may contribute to the neurological phenotype of cerebellar ataxia and that targeting GABAA receptors might represent a feasible complementary strategy to treat cerebellar ataxia.

Canine Mammary Tumours Are Affected by Frequent Copy Number Aberrations, including Amplification of MYC and Loss of PTEN.

BACKGROUND: Copy number aberrations frequently occur during the development of many cancers. Such events affect dosage of involved genes and may cause further genomic instability and progression of cancer. In this survey, canine SNP microarrays were used to study 117 canine mammary tumours from 69 dogs. RESULTS: We found a high occurrence of copy number aberrations in canine mammary tumours, losses being more frequent than gains. Increased frequency of aberrations and loss of heterozygosity were positively correlated with increased malignancy in terms of histopathological diagnosis. One of the most highly recurrently amplified regions harbored the MYC gene. PTEN was located to a frequently lost region and also homozygously deleted in five tumours. Thus, deregulation of these genes due to copy number aberrations appears to be an important event in canine mammary tumour development. Other potential contributors to canine mammary tumour pathogenesis are COL9A3, INPP5A, CYP2E1 and RB1. The present study also shows that a more detailed analysis of chromosomal aberrations associated with histopathological parameters may aid in identifying specific genes associated with canine mammary tumour progression. CONCLUSIONS: The high frequency of copy number aberrations is a prominent feature of canine mammary tumours as seen in other canine and human cancers. Our findings share several features with corresponding studies in human breast tumours and strengthen the dog as a suitable model organism for this disease.

Distribution and function of polycystin-2 in mouse retinal ganglion cells.

The polycystin family of transient receptor potential (TRP) channels form Ca(2+) regulated cation channels with distinct subcellullar localizations and functions. As part of heteromultimeric channels and multi-protein complexes, polycystins control intracellular Ca(2+) signals and more generally the translation of extracellular signals and stimuli to intracellular responses. Polycystin-2 channels have been cloned from retina, but their distribution and function in retinal ganglion cells (RGCs) have not yet been established. In the present study, we determined cellular and subcellular localization as well as functional properties of polycystin-2 channels in RGCs. Polycystin-2 expression and distribution in RGCs was assessed by immunohistochemistry on vertical cryostat section of mouse retina as well as primary cultured mouse RGCs, using fluorescence microscopy. Biophysical and pharmacological properties of polycystin-2 channels isolated from primary cultured RGCs were determined using planar lipid bilayer electrophysiology. We detected polycystin-2 immunoreactivity both in the ganglion cell layer as well as in primary cultured RGCs. Subcellular analysis revealed strong cytosolic localization pattern of polycystin-2. Polycystin-2 channel current was Ca(2+) activated, had a maximum slope conductance of 114 pS, and could be blocked in a dose-dependent manner by increasing concentrations of Mg(2+). The cytosolic localization of polycystin-2 in RGCs is in accordance with its function as intracellular Ca(2+) release channel. We conclude that polycystin-2 forms functional channels in RGCs, of which biophysical and pharmacological properties are similar to polycystin-2 channels reported for other tissues and organisms. Our data suggest a potential role for polycystin-2 in RGC Ca(2+) signaling.

Novel mechanism of increased Ca2+ release following oxidative stress in neuronal cells involves type 2 inositol-1,4,5-trisphosphate receptors.

Dysregulation of Ca(2+) signaling following oxidative stress is an important pathophysiological mechanism of many chronic neurodegenerative disorders, including Alzheimer's disease, age-related macular degeneration, glaucomatous and diabetic retinopathies. However, the underlying mechanisms of disturbed intracellular Ca(2+) signaling remain largely unknown. We here describe a novel mechanism for increased intracellular Ca(2+) release following oxidative stress in a neuronal cell line. Using an experimental approach that included quantitative polymerase chain reaction, quantitative immunoblotting, microfluorimetry and the optical imaging of intracellular Ca(2+) release, we show that sub-lethal tert-butyl hydroperoxide-mediated oxidative stress result in a selective up-regulation of type-2 inositol-1,4,5,-trisphophate receptors. This oxidative stress mediated change was detected both at the transcriptional and translational level and functionally resulted in increased Ca(2+) release into the nucleoplasm from the membranes of the nuclear envelope at a given receptor-specific stimulus. Our data describe a novel source of Ca(2+) dysregulation induced by oxidative stress with potential relevance for differential subcellular Ca(2+) signaling specifically within the nucleus and the development of novel neuroprotective strategies in neurodegenerative disorders.

Intracellular mechanisms of N-acylethanolamine-mediated neuroprotection in a rat model of stroke.

N-acyl ethanolamines (NAEs) are endogenous lipids that are synthesized in response to tissue injury, including ischemia and stroke, suggesting they may exhibit neuroprotective properties. We hypothesized that NAE 16:0 (palmitoylethanolamine) is neuroprotective against ischemia-reperfusion injury in rats, a widely employed model of stroke, and that neuroprotection is mediated through an intracellular mechanism independent of known NAE receptors. Administration of NAE 16:0 from 30 min before to 2 h after stroke significantly reduced cortical and subcortical infarct volume, and correlated with an improvement of the neurological phenotype, as assessed by the neurological deficit score. We here show that NAE 16:0-mediated neuroprotection was independent of cannabinoid (CB1) and vanilloid (VR1) receptor activation, known NAE receptors on the plasma membrane, as determined by inclusion of specific inhibitors. The inclusion of an NAE uptake inhibitor (AM404), however, completely reversed NAE 16:0-mediated neuroprotection, suggesting that NAE 16:0s effects are through an intracellular mechanism. NAE 16:0 produced a significant reduction in the number of cells undergoing apoptosis and reversed ischemia-induced upregulation of several proteins, including inducible nitric oxide synthase and transcription factor NFkappaB. Our findings suggest that NAE 16:0-mediated neuroprotection is due to the reduction of neuronal apoptosis and inflammation in the brain.

Aberrant cerebellar granule cell-specific GABAA receptor expression in the epileptic and ataxic mouse mutant, Tottering.

The Tottering (cacna1a(tg)) mouse arose as a consequence of a spontaneous mutation in cacna1a, the gene encoding the pore-forming subunit of the pre-synaptic P/Q-type voltage-gated calcium channel (VGCC, Ca(V)2.1). The mouse phenotype includes ataxia and intermittent myoclonic seizures which have been attributed to impaired excitatory neurotransmission at cerebellar granule cell (CGC) parallel fiber-Purkinje cell (PF-PC) synapses [Zhou YD, Turner TJ, Dunlap K (2003) Enhanced G-protein-dependent modulation of excitatory synaptic transmission in the cerebellum of the Ca(2+)-channel mutant mouse, tottering. J Physiol 547:497-507]. We hypothesized that the expression of cerebellar GABA(A) receptors may be affected by the mutation. Indeed, abnormal GABA(A) receptor function and expression in the cacna1a(tg) forebrain has been reported previously [Tehrani MH, Barnes EM Jr (1995) Reduced function of gamma-aminobutyric acid A receptors in tottering mouse brain: role of cAMP-dependent protein kinase. Epilepsy Res 22:13-21; Tehrani MH, Baumgartner BJ, Liu SC, Barnes EM Jr (1997) Aberrant expression of GABA(A) receptor subunits in the tottering mouse: an animal model for absence seizures. Epilepsy Res 28:213-223]. Here we show a deficit of 40.2+/-3.6% in the total number of cerebellar GABA(A) receptors expressed (gamma2+delta subtypes) in adult cacna1a(tg) relative to controls. [(3)H]Muscimol autoradiography identified that this was partly due to a significant loss of CGC-specific alpha6 subunit-containing GABA(A) receptor subtypes. A large proportion of this loss of alpha6 receptors was attributable to a significantly reduced expression of the CGC-specific benzodiazepine-insensitive Ro15-4513 (BZ-IS) binding subtype, alpha6betagamma2 subunit-containing receptors. BZ-IS binding was reduced by 36.6+/-2.6% relative to controls in cerebellar membrane homogenates and by 37.2+/-3.7% in cerebellar sections. Quantitative immunoblotting revealed that the steady-state expression level of alpha6 and gamma2 subunits was selectively reduced relative to controls by 30.2+/-8.2% and 38.8+/-13.1%, respectively, alpha1, beta3 and delta were unaffected. Immunohistochemically probed control and cacna1a(tg) cerebellar sections verified that alpha6 and gamma2 subunit expression was reduced and that this deficit was restricted to the CGC layer. Thus, we have shown that abnormal cerebellar P/Q-type VGCC activity results in a deficit of CGC-specific subtype(s) of GABA(A) receptors which may contribute to, or may be a consequence of the impaired cerebellar network signaling that occurs in cacna1a(tg) mice.

Characterization of acetylcholine release and the compensatory contribution of non-Ca(v)2.1 channels at motor nerve terminals of leaner Ca(v)2.1-mutant mice.

The severely ataxic and epileptic mouse leaner (Ln) carries a natural splice site mutation in Cacna1a, leading to a C-terminal truncation of the encoded Ca(v)2.1 alpha(1) protein. Ca(v)2.1 is a neuronal Ca(2+) channel, mediating neurotransmitter release at many central synapses and the peripheral neuromuscular junction (NMJ). With electrophysiological analyses we demonstrate severely reduced ( approximately 50%) neurotransmitter release at Ln NMJs. This equals the reduction at NMJs of Cacna1a null-mutant (Ca(v)2.1-KO) mice, which display a neurological phenotype remarkably similar to that of Ln mice. However, using selective Ca(v) channel blocking compounds we revealed a compensatory contribution profile of non-Ca(v)2.1 type channels at Ln NMJs that differs completely from that at Ca(v)2.1-KO NMJs. Our data indicate that the residual function and presence of Ln-mutated Ca(v)2.1 channels precludes presynaptic compensatory recruitment of Ca(v)1 and Ca(v)2.2 channels, and hampers that of Ca(v)2.3 channels. This is the first report directly showing at single synapses the deficits and plasticity in transmitter release resulting from the Ln mutation of Cacna1a.

Gene dosage-dependent transmitter release changes at neuromuscular synapses of CACNA1A R192Q knockin mice are non-progressive and do not lead to morphological changes or muscle weakness.

Ca(v)2.1 channels mediate neurotransmitter release at the neuromuscular junction (NMJ) and at many central synapses. Mutations in the encoding gene, CACNA1A, are thus likely to affect neurotransmitter release. Previously, we generated mice carrying the R192Q mutation, associated with human familial hemiplegic migraine type-1, and showed first evidence of enhanced presynaptic Ca(2+) influx [Neuron 41 (2004) 701]. Here, we characterize transmitter release in detail at mouse R192Q NMJs, including possible gene-dosage dependency, progression of changes with age, and associated morphological damage and muscle weakness. We found, at low Ca(2+), decreased paired-pulse facilitation of evoked acetylcholine release, elevated release probability, and increased size of the readily releasable transmitter vesicle pool. Spontaneous release was increased over a broad range of Ca(2+) concentrations (0.2-5mM). Upon high-rate nerve stimulation we observed some extra rundown of transmitter release. However, no clinical evidence of transmission block or muscle weakness was found, assessed with electromyography, grip-strength testing and muscle contraction experiments. We studied both adult ( approximately 3-6 months-old) and aged ( approximately 21-26 months-old) R192Q knockin mice to assess effects of chronic elevation of presynaptic Ca(2+) influx, but found no additional or progressive alterations. No changes in NMJ size or relevant ultrastructural parameters were found, at either age. Our characterizations strengthen the hypothesis of increased Ca(2+) flux through R192Q-mutated presynaptic Ca(v)2.1 channels and show that the resulting altered neurotransmitter release is not associated with morphological changes at the NMJ or muscle weakness, not even in the longer term.

Measurement of liver blood flow using oxygen-15 labelled water and dynamic positron emission tomography: limitations of model description.

To date no satisfactory method has been available for the quantitative in vivo measurement of the complex hepatic blood flow. In this study two modelling approaches are proposed for the analysis of liver blood flow using positron emission tomography (PET). Five experiments were performed on three foxhounds. The anaesthetised dogs were each given an intravenous bolus injection of oxygen-15 labelled water, and their livers were then scanned using PET. Radioactivity in the blood from the aorta and portal vein was measured directly and simultaneously using closed external circuits. Time-activity curves were constructed from sequential PET data. Data analysis was performed by assuming that water behaves as a freely diffusible tracer and adapting the standard one-compartment blood flow model to describe the dual blood supply of the liver. Two particular modelling approaches were investigated: the dual-input model used both directly measured input functions (i.e. using the hepatic artery and the portal vein input, determined from the radioactivity detected in the aorta and portal vein respectively) whereas the single-input model used only the measured arterial curve and predicted the corresponding portal input function. Hepatic arterial flow, portal flow and blood volume were fitted from the PET data in several regions of the liver. The resulting estimates were then compared with reference blood flow measurements, obtained using a standard microsphere technique. The microspheres were injected in a separate experiment on the same dogs immediately prior to PET scanning. Whilst neither the single- nor the dual-input models accurately reproduced the arterial reference flow values, the flow values from the single-input model were closer to the microsphere flow values. The proposed single-input model would be a good approximation for liver blood flow measurements in man. The observed discrepancies between the PET and microsphere flow values may be due to the inherent temporal and spatial heterogeneity of liver blood flow. The results presented suggest that adaptation of the standard one-compartment blood flow model to describe the dual blood supply of the liver is limited and other flow tracers have to be considered for quantitative PET measurements in the liver.