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Introduction: Diabetes often leads to lower urinary tract dysfunction. The most frequently assessed parameter of urinary bladder dysfunction in animal models of diabetes is an enlargement of the bladder, which is consistently observed in type 1 and less consistently in type 2 diabetes. The vast majority of studies on bladder weight in animal models of diabetes and obesity has been performed in males, and no studies have directly compared this outcome parameter between sexes. Methods: Therefore, we have compared bladder weight and bladder/body weight ratio in five mouse models of obesity and diabetes (RIP-LCMV, db/db, ob/ob (two studies), insulin receptor substrate 2 (IRS2) knock-out mice and mice on a high-fat diet; pre-specified secondary analysis of a previously reported study). Results: In a pooled analysis of the control groups of all studies, females exhibited slightly lower glucose levels, lower body weight, and lower bladder weight, but bladder/body weight ratio was similar in both sexes (0.957 vs. 0.986 mg/g, mean difference 0.029 [−0.06; 0.118]). Among the six diabetic/obese groups, bladder/body weight ratio was similar in both sexes in three but smaller in female mice in three other groups. The mRNA expression of a panel of genes implied in the pathophysiology of bladder enlargement and/or fibrosis and inflammation did not differ systematically between sexes. Conclusions: We conclude that sex differences in diabetes/obesity-associated bladder enlargement may be model dependent.

Mechanisms of [Ca 2+ ] i Transient Decrease in Cardiomyopathy of db / db Type 2 Diabetic Mice Laetitia Pereira 1 , Jan Matthes 2 , Iris Schuster 3 , Héctor H. Valdivia 4 , Stefan Herzig 2 , Sylvain Richard 1 and Ana M. Gómez 1 1 Institut National de la Santé et de la Recherche Médicale, U 637, University of Montpellier 1, Montpellier, France 2 Department of Pharmacology and Center of Molecular Medicine, University of Cologne, Cologne, Germany 3 School of Medicine, University of Montpellier 1, EA 2992, Nîmes, France 4 Department of Physiology, University of Wisconsin Medical School, Madison, Wisconsin Address correspondence and reprint requests to Ana María Gómez, Institut National de la Santé et de la Recherche Médicale U-637, CHU A. de Villeneuve, 34295 Montpellier, France. E-mail: agomez{at}montp.inserm.fr Abstract Cardiovascular disease is the leading cause of death in the diabetic population. However, molecular mechanisms underlying diabetic cardiomyopathy remain unclear. We analyzed Ca 2+ -induced Ca 2+ release and excitation-contraction coupling in db / db obese type 2 diabetic mice and their control littermates. Echocardiography showed a systolic dysfunction in db / db mice. Two-photon microscopy identified intracellular calcium concentration ([Ca 2+ ] i ) transient decrease in cardiomyocytes within the whole heart, which was also found in isolated myocytes by confocal microscopy. Global [Ca 2+ ] i transients are constituted of individual Ca 2+ sparks. Ca 2+ sparks in db / db cardiomyocytes were less frequent than in +/+ myocytes, partly because of a depression in sarcoplasmic reticulum Ca 2+ load but also because of a reduced expression of ryanodine receptor Ca 2+ channels (RyRs), revealed by [ 3 H]ryanodine binding assay. Ca 2+ efflux through Na + /Ca 2+ exchanger was increased in db / db myocytes. Calcium current, I Ca , triggers sarcoplasmic reticulum Ca 2+ release and is also involved in sarcoplasmic reticulum Ca 2+ refilling. Macroscopic I Ca was reduced in db / db cells, but single Ca 2+ channel activity was similar, suggesting that diabetic myocytes express fewer functional Ca 2+ channels, which was confirmed by Western blots. These results demonstrate that db / db mice show depressed cardiac function, at least in part, because of a general reduction in the membrane permeability to Ca 2+ . As less Ca 2+ enters the cell through I Ca , less Ca 2+ is released through RyRs. [Ca2+]i, intracellular calcium concentration NCX, Na+/Ca2+ exchanger RyR, ryanodine receptor Ca2+ channel SERCA, sarcoplasmic reticulum Ca2+ ATPase Footnotes The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Accepted November 30, 2005. Received October 3, 2005. DIABETES

Various strategies have been published enabling cardiomyocyte differentiation of human induced pluripotent stem (iPS) cells. However the complex nature of signaling pathways involved as well as line-to-line variability compromises the application of a particular protocol to robustly obtain cardiomyocytes from multiple iPS lines. Hence it is necessary to identify optimized protocols with alternative combinations of specific growth factors and small molecules to enhance the robustness of cardiac differentiation. Here we focus on systematic modulation of BMP and WNT signaling to enhance cardiac differentiation. Moreover, we improve the efficacy of cardiac differentiation by enrichment via lactate. Using our protocol we show efficient derivation of cardiomyocytes from multiple human iPS lines. In particular we demonstrate cardiomyocyte differentiation within 15 days with an efficiency of up to 95 % as judged by flow cytometry staining against cardiac troponin T. Cardiomyocytes derived were functionally validated by alpha-actinin staining, transmission electron microscopy as well as electrophysiological analysis. We expect our protocol to provide a robust basis for scale-up production of functional iPS cell-derived cardiomyocytes that can be used for cell replacement therapy and disease modeling.

Autism Spectrum Disorders (ASD) are complex neurodevelopmental diseases clinically defined by dysfunction of social interaction. Dysregulation of cellular calcium homeostasis might be involved in ASD pathogenesis, and genes coding for the L-type calcium channel subunits CaV1.2 (CACNA1C) and CaVβ2 (CACNB2) were recently identified as risk loci for psychiatric diseases. Here, we present three rare missense mutations of CACNB2 (G167S, S197F, and F240L) found in ASD-affected families, two of them described here for the first time (G167S and F240L). All these mutations affect highly conserved regions while being absent in a sample of ethnically matched controls. We suggest the mutations to be of physiological relevance since they modulate whole-cell Ba2+ currents through calcium channels when expressed in a recombinant system (HEK-293 cells). Two mutations displayed significantly decelerated time-dependent inactivation as well as increased sensitivity of voltage-dependent inactivation. In contrast, the third mutation (F240L) showed significantly accelerated time-dependent inactivation. By altering the kinetic parameters, the mutations are reminiscent of the CACNA1C mutation causing Timothy Syndrome, a Mendelian disease presenting with ASD. In conclusion, the results of our first-time biophysical characterization of these three rare CACNB2 missense mutations identified in ASD patients support the hypothesis that calcium channel dysfunction may contribute to autism.

Although doctor-patient communication is essential for drug prescription, the literature reveals deficits in this area. An educational approach at the Cologne medical faculty aims at identifying and addressing those deficits in medical students. Fifth-year medical students first conducted a simulated prescription talk spontaneously. Subsequently, the conversation was discussed with peer students. A pharmacist moderated the discussion based upon a previously developed conversation guide. Afterwards, the same student had the conversation again, but as if for the first time. Conversations were video-recorded, transcribed and subjected to quantitative content analysis. Four days after the simulation, the students who conducted the talk, those who observed and discussed it, and students who did neither, completed a written test that focused on the content of an effective prescription talk. Content analysis revealed clear deficits in spontaneously led prescription talks. Even essential information as on adverse drug reactions were often lacking. Prescription talks became clearly more informative and comprehensive after the short, guided peer discussion. With regard to a comprehensive, informative prescription talk, the written test showed that both the students who conducted the talk and those who only observed it performed clearly better than the students who did not participate in the educational approach. Deficits regarding prescription talks are present in 5 th year medical students. We provide an approach to both identify and address these deficits. It thus may be an example for training medical students in simulated and clinical environments like the EACPT recommended to improve pharmacology education.

Patients want more information and active participation in medical decisions. Information and active participation correlate with increased adherence. A conversation guide, combining patient-relevant drug information with steps of shared decision-making, was developed to support physicians in effective and efficient prescription talks. Six GP trainees in community-based primary care practices participated in a controlled pilot study in sequential pre-post design. Initially, they conducted 41 prescription talks as usual, i.e., without knowing the guide. Then, they conducted 23 talks considering the guide (post-intervention phase). Immediately after the respective talk, patients filled in a questionnaire on satisfaction with the information on medication and physician–patient interaction, and physicians about their satisfaction with the talk and the application of the guide. Patients felt better informed after guide-based prescription talks (e.g., SIMS-D in median 10 vs. 17, p < 0.05), more actively involved (KPF-A for patient activation 2.9 ± 0.8 vs. 3.6 ± 0.8, p < 0.05), and more satisfied with the physician–patient interaction. Physicians rated the guide helpful and feasible. Their satisfaction with the conversation was significantly enhanced during the post-intervention phase. The evaluation of the duration of the talk was not influenced. Enhanced patients’ and physicians’ satisfaction with prescription talks encourages further examinations of the conversation guide. We invite physicians to try our guide in everyday medical practice.

Purpose Poor medication communication of physicians to patients is detrimental, e.g. for medication adherence. Reasons for physicians’ deficits in medication communication may be unfavourable conditions in daily practice or already insufficient training during their (undergraduate) medical studies. We explored medical students’ communication on new medications in simulated physician-patient conversations to identify actual deficits indicating apparent educational needs. Methods Fifth year medical students attending a mandatory course at the University of Cologne had simulated physician-patient consultations aiming at drug prescription. In 2015, 21 consultations were recorded, transcribed and subjected to qualitative content analysis based on the method of inductive coding. Results Even essential information on drug therapy was often lacking (e.g. adverse effects, drug administration). Some aspects were addressed more frequently than others. This seemed to differ depending on the diagnosis underlying the particular treatment (acute event vs. chronic disease). The extent of information on drug treatments given in simulated physician-patient consultations varied significantly between students. Conclusions Fifth year medical students showed appreciable deficits in communicating drug prescriptions to patients though there were remarkable inter-individual differences. Our findings suggest that communication on drug therapy to patients is no self-evolving skill. Thus, there is obviously a need for emphasizing medication communication in the training of medical students. Communication aids specifically aiming at medication communication might facilitate learning of adequate medication communication skills.

Two pertussis toxin sensitive G(i) proteins, G(i2) and G(i3), are expressed in cardiomyocytes and upregulated in heart failure. It has been proposed that the highly homologous G(i) isoforms are functionally distinct. To test for isoform-specific functions of G(i) proteins, we examined their role in the regulation of cardiac L-type voltage-dependent calcium channels (L-VDCC). Ventricular tissues and isolated myocytes were obtained from mice with targeted deletion of either Gα(i2) (Gα(i2) (-/-)) or Gα(i3) (Gα(i3) (-/-)). mRNA levels of Gα(i/o) isoforms and L-VDCC subunits were quantified by real-time PCR. Gα(i) and Ca(v)α(1) protein levels as well as protein kinase B/Akt and extracellular signal-regulated kinases 1/2 (ERK1/2) phosphorylation levels were assessed by immunoblot analysis. L-VDCC function was assessed by whole-cell and single-channel current recordings. In cardiac tissue from Gα(i2) (-/-) mice, Gα(i3) mRNA and protein expression was upregulated to 187 ± 21% and 567 ± 59%, respectively. In Gα(i3) (-/-) mouse hearts, Gα(i2) mRNA (127 ± 5%) and protein (131 ± 10%) levels were slightly enhanced. Interestingly, L-VDCC current density in cardiomyocytes from Gα(i2) (-/-) mice was lowered (-7.9 ± 0.6 pA/pF, n = 11, p<0.05) compared to wild-type cells (-10.7 ± 0.5 pA/pF, n = 22), whereas it was increased in myocytes from Gα(i3) (-/-) mice (-14.3 ± 0.8 pA/pF, n = 14, p<0.05). Steady-state inactivation was shifted to negative potentials, and recovery kinetics slowed in the absence of Gα(i2) (but not of Gα(i3)) and following treatment with pertussis toxin in Gα(i3) (-/-). The pore forming Ca(v)α(1) protein level was unchanged in all mouse models analyzed, similar to mRNA levels of Ca(v)α(1) and Ca(v)β(2) subunits. Interestingly, at the cellular signalling level, phosphorylation assays revealed abolished carbachol-triggered activation of ERK1/2 in mice lacking Gα(i2). Our data provide novel evidence for an isoform-specific modulation of L-VDCC by Gα(i) proteins. In particular, loss of Gα(i2) is reflected by alterations in channel kinetics and likely involves an impairment of the ERK1/2 signalling pathway.

Increased activity of single ventricular L-type Ca(2+)-channels (L-VDCC) is a hallmark in human heart failure. Recent findings suggest differential modulation by several auxiliary beta-subunits as a possible explanation. By molecular and functional analyses of human and murine ventricles, we find that enhanced L-VDCC activity is accompanied by altered expression pattern of auxiliary L-VDCC beta-subunit gene products. In HEK293-cells we show differential modulation of single L-VDCC activity by coexpression of several human cardiac beta-subunits: Unlike beta(1) or beta(3) isoforms, beta(2a) and beta(2b) induce a high-activity channel behavior typical of failing myocytes. In accordance, beta(2)-subunit mRNA and protein are up-regulated in failing human myocardium. In a model of heart failure we find that mice overexpressing the human cardiac Ca(V)1.2 also reveal increased single-channel activity and sarcolemmal beta(2) expression when entering into the maladaptive stage of heart failure. Interestingly, these animals, when still young and non-failing (\"Adaptive Phase\"), reveal the opposite phenotype, viz: reduced single-channel activity accompanied by lowered beta(2) expression. Additional evidence for the cause-effect relationship between beta(2)-subunit expression and single L-VDCC activity is provided by newly engineered, double-transgenic mice bearing both constitutive Ca(V)1.2 and inducible beta(2) cardiac overexpression. Here in non-failing hearts induction of beta(2)-subunit overexpression mimicked the increase of single L-VDCC activity observed in murine and human chronic heart failure. Our study presents evidence of the pathobiochemical relevance of beta(2)-subunits for the electrophysiological phenotype of cardiac L-VDCC and thus provides an explanation for the single L-VDCC gating observed in human and murine heart failure.

Two subtypes of β-adrenoceptors, β 1 and β 2 , mediate cardiac catecholamine effects. These two types differ qualitatively, e.g., regarding G protein coupling and calcium channel stimulation. Transgenic mice overexpressing human β 2 -adrenoceptors survive high-expression levels, unlike mice overexpressing β 1 -adrenoceptors. We examined the role of inhibitory G i proteins, known to be activated by β 2 - but not β 1 -adrenoceptors, on the chronic effects of human β 2 -adrenoreceptor overexpression in transgenic mice. These mice were crossbred with mice where Gα i2 , a functionally important cardiac G i α-subunit, was inactivated by targeted gene deletion. Survival of β 2 -adrenoreceptor transgenic mice was reduced by heterozygous inactivation of Gα i2 . Homozygous knockout/β 2 -adrenoreceptor transgenic mice died within 4 days after birth. Heterozygous knockout/β 2 -adrenoreceptor transgenic mice developed more pronounced cardiac hypertrophy and earlier heart failure compared with β 2 -adrenoreceptor transgenic mice. Single calcium-channel activity was strongly suppressed in heterozygous knockout/β 2 -adrenoreceptor transgenic mice. In cardiomyocytes from these mice, pertussis toxin treatment in vitro fully restored channel activity and enhanced channel activity in cells from homozygous Gα i2 knockout animals. Cardiac Gα i3 protein was increased in all Gα i2 knockout mouse strains. Our results demonstrate that Gα i2 takes an essential protective part in chronic signaling of overexpressed β 2 -adrenoceptors, leading to prolonged survival and delayed cardiac pathology. However, reduction of calcium-channel activity by β 2 -adrenoreceptor overexpression is due to a different pertussis-toxin-sensitive pathway, most likely by Gα i3 . This result indicates that subtype-specific signaling of β 2 -adrenoreceptor functionally bifurcates at the level of G i , leading to different effects depending on the Gα isoform. L-type calcium channel single channel recording mouse genetics survival curve pertussis toxin