Action potential variability in human pluripotent stem cell-derived cardiomyocytes obtained from healthy donors

Human pluripotent stem cells (PSC) have been used for disease modelling, after differentiation into the desired cell type. Electrophysiologic properties of cardiomyocytes derived from pluripotent stem cells are extensively used to model cardiac arrhythmias, in cardiomyopathies and channelopathies. This requires strict control of the multiple variables that can influence the electrical properties of these cells. In this article, we report the action potential variability of 780 cardiomyocytes derived from pluripotent stem cells obtained from six healthy donors. We analyze the overall distribution of action potential (AP) data, the distribution of action potential data per cell line, per differentiation protocol and batch. This analysis indicates that even using the same cell line and differentiation protocol, the differentiation batch still affects the results. This variability has important implications in modeling arrhythmias and imputing pathogenicity to variants encountered in patients with arrhythmic diseases. We conclude that even when using isogenic cell lines to ascertain pathogenicity to variants associated to arrythmias one should use cardiomyocytes derived from pluripotent stem cells using the same differentiation protocol and batch and pace the cells or use only cells that have very similar spontaneous beat rates. Otherwise, one may find phenotypic variability that is not attributable to pathogenic variants

R534C mutation in hERG causes a trafficking defect in iPSC-derived cardiomyocytes from patients with type 2 long QT syndrome

Patient-specific cardiomyocytes obtained from induced pluripotent stem cells (CM-iPSC) offer unprecedented mechanistic insights in the study of inherited cardiac diseases. The objective of this work was to study a type 2 long QT syndrome (LQTS2)-associated mutation (c.1600C > T in KCNH2, p.R534C in hERG) in CM-iPSC. Peripheral blood mononuclear cells were isolated from two patients with the R534C mutation and iPSCs were generated. In addition, the same mutation was inserted in a control iPSC line by genome editing using CRISPR/Cas9. Cells expressed pluripotency markers and showed spontaneous differentiation into the three embryonic germ layers. Electrophysiology demonstrated that action potential duration (APD) of LQTS2 CM-iPSC was significantly longer than that of the control line, as well as the triangulation of the action potentials (AP), implying a longer duration of phase 3. Treatment with the IKr inhibitor E4031 only caused APD prolongation in the control line. Patch clamp showed a reduction of IKr on LQTS2 CM-iPSC compared to control, but channel activation was not significantly affected. Immunofluorescence for hERG demonstrated perinuclear staining in LQTS2 CM-iPSC. In conclusion, CM-iPSC recapitulated the LQTS2 phenotype and our findings suggest that the R534C mutation in KCNH2 leads to a channel trafficking defect to the plasma membrane.Fil: Mesquita, Fernanda C. P.. Universidade Federal do Rio de Janeiro; BrasilFil: Arantes, Paulo C.. Universidade Federal do Rio de Janeiro; BrasilFil: Kasai Brunswick, Tais H.. Universidade Federal do Rio de Janeiro; BrasilFil: Araujo, Dayana S.. Universidade Federal do Rio de Janeiro; BrasilFil: Gubert, Fernanda. Universidade Federal do Rio de Janeiro; BrasilFil: Monnerat, Gustavo. Universidade Federal do Rio de Janeiro; BrasilFil: Silva dos Santos, Danúbia. Universidade Federal do Rio de Janeiro; BrasilFil: Neiman, Gabriel. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Fundación para la Lucha contra las Enfermedades Neurológicas de la Infancia; ArgentinaFil: Leitão, Isabela C.. Universidade Federal do Rio de Janeiro; BrasilFil: Barbosa, Raiana A. Q.. Universidade Federal do Rio de Janeiro; BrasilFil: Coutinho, Jorge L.. National Institute Of Cardiology; BrasilFil: Vaz, Isadora M.. Pontificia Universidad Catolica de Parana; BrasilFil: dos Santos, Marcus N.. Universidade Federal do Rio de Janeiro; BrasilFil: Borgonovo, Tamara. Pontificia Universidad Catolica de Parana; BrasilFil: Cruz, Fernando E. S.. National Institute of Cardiology; BrasilFil: Miriuka, Santiago Gabriel. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Fundación para la Lucha contra las Enfermedades Neurológicas de la Infancia; ArgentinaFil: Medei, Emiliano H.. Universidade Federal do Rio de Janeiro; BrasilFil: Campos de Carvalho, Antonio C.. Universidade Federal do Rio de Janeiro; Brasil. National Institute of Cardiology; Brasil. National Institute for Science and Technology in Regenerative Medicine; BrasilFil: Carvalho, Adriana B.. Universidade Federal do Rio de Janeiro; Brasil. National Institute for Science and Technology in Regenerative Medicine; Brasi

E-service e performance aziendali

La tecnologia digitale e le performance aziendal

Toll-like Receptor 4 Activation Promotes Cardiac Arrhythmias By Decreasing The Transient Outward Potassium Current (ito) Through An Irf3-dependent And Myd88-independent Pathway

Cardiac arrhythmias are one of the main causes of death worldwide. Several studies have shown that inflammation plays a key role in different cardiac diseases and Toll-like receptors (TLRs) seem to be involved in cardiac complications. In the present study, we investigated whether the activation of TLR4 induces cardiac electrical remodeling and arrhythmias, and the signaling pathway involved in these effects. Membrane potential was recorded in Wistar rat ventricle. Ca2+ transients, as well as the L-type Ca2+ current (ICaL) and the transient outward K+ current (Ito), were recorded in isolated myocytes after 24h exposure to the TLR4 agonist, lipopolysaccharide (LPS, 1μg/ml). TLR4 stimulation in vitro promoted a cardiac electrical remodeling that leads to action potential prolongation associated with arrhythmic events, such as delayed afterdepolarization and triggered activity. After 24h LPS incubation, Ito amplitude, as well as Kv4.3 and KChIP2 mRNA levels were reduced. The Ito decrease by LPS was prevented by inhibition of interferon regulatory factor 3 (IRF3), but not by inhibition of interleukin-1 receptor-associated kinase 4 (IRAK4) or nuclear factor kappa B (NF-κB). Extrasystolic activity was present in 25% of the cells, but apart from that, Ca2+ transients and ICaL were not affected by LPS; however, Na+/Ca2+ exchanger (NCX) activity was apparently increased. We conclude that TLR4 activation decreased Ito, which increased AP duration via a MyD88-independent, IRF3-dependent pathway. The longer action potential, associated with enhanced Ca2+ efflux via NCX, could explain the presence of arrhythmias in the LPS group.767075Murray, C.J., Lopez, A.D., Alternative projections of mortality and disability by cause 1990-2020: Global Burden of Disease Study (1997) Lancet, 349, pp. 1498-1504Randomised trial of cholesterol lowering in 4444 patients with coronary heart disease: the Scandinavian Simvastatin Survival Study (4S) (1994) Lancet, 344, pp. 1383-1389Algra, A., Tijssen, J.G., Roelandt, J.R., Pool, J., Lubsen, J., QTc prolongation measured by standard 12-lead electrocardiography is an independent risk factor for sudden death due to cardiac arrest (1991) Circulation, 83, pp. 1888-1894Casis, O., Gallego, M., Iriarte, M., Sánchez-Chapula, J.A., Effects of diabetic cardiomyopathy on regional electrophysiologic characteristics of rat ventricle (2000) Diabetologia, 43, pp. 101-109Kang, Y.J., Cardiac hypertrophy: a risk factor for QT-prolongation and cardiac sudden death (2006) Toxicol Pathol, 34, pp. 58-66Leurs, R., Church, M.K., Taglialatela, M., H1-antihistamines: inverse agonism, anti-inflammatory actions and cardiac effects (2002) Clin Exp Allergy, 32, pp. 489-498Monnerat-Cahli, G., Trentin-Sonoda, M., Guerra, B., Manso, G., Ferreira, A.C., Silva, D.L., Bone marrow mesenchymal stromal cells rescue cardiac function in streptozotocin-induced diabetic rats (2013) Int J Cardiol, 171, pp. 199-208Danesh, J., Wheeler, J.G., Hirschfield, G.M., Eda, S., Eiriksdottir, G., Rumley, A., C-reactive protein and other circulating markers of inflammation in the prediction of coronary heart disease (2004) N Engl J Med, 350, pp. 1387-1397Scheruebel, S., Koyani, C.N., Hallström, S., Lang, P., Platzer, D., Mächler, H., If blocking potency of ivabradine is preserved under elevated endotoxin levels in human atrial myocytes (2014) J Mol Cell Cardiol, 72, pp. 64-73Devaraj, S., Dasu, M.R., Rockwood, J., Winter, W., Griffen, S.C., Jialal, I., Increased toll-like receptor (TLR) 2 and TLR4 expression in monocytes from patients with type 1 diabetes: further evidence of a proinflammatory state (2008) J Clin Endocrinol Metab, 93, pp. 578-583Casey, L.C., Balk, R.A., Bone, R.C., Plasma cytokine and endotoxin levels correlate with survival in patients with the sepsis syndrome (1993) Ann Intern Med, 119, pp. 771-778Nozaki, N., Shishido, T., Takeishi, Y., Kubota, I., Modulation of doxorubicin-induced cardiac dysfunction in toll-like receptor-2-knockout mice (2004) Circulation, 110, pp. 2869-2874Shishido, T., Nozaki, N., Yamaguchi, S., Shibata, Y., Nitobe, J., Miyamoto, T., Toll-like receptor-2 modulates ventricular remodeling after myocardial infarction (2003) Circulation, 108, pp. 2905-2910Fallach, R., Shainberg, A., Avlas, O., Fainblut, M., Chepurko, Y., Porat, E., Cardiomyocyte Toll-like receptor 4 is involved in heart dysfunction following septic shock or myocardial ischemia (2010) J Mol Cell Cardiol, 48, pp. 1236-1244Panama, B.K., Latour-Villamil, D., Farman, G.P., Zhao, D., Bolz, S.-S., Kirshenbaum, L.A., Nuclear factor κB downregulates the transient outward potassium current Ito, f through control of KChIP2 expressionnovelty and significance (2011) Circ Res, 108, pp. 537-543Medei, E.H., Nascimento, J.H., Pedrosa, R.C., Barcellos, L., Masuda, M.O., Sicouri, S., Antibodies with beta-adrenergic activity from chronic chagasic patients modulate the QT interval and M cell action potential duration (2008) Europace, 10, pp. 868-876Penna, L.B., Bassani, R.A., Increased spontaneous activity and reduced inotropic response to catecholamines in ventricular myocytes from footshock-stressed rats (2010) Stress, 13, pp. 73-82Bassani, R.A., Ricardo, R.A., Bassani, J.W., Estimation of the fractional sarcoplasmic reticulum Ca2+ release in intact cardiomyocytes using integrated Ca2+ fluxes (2012) Gen Physiol Biophys, 31, pp. 401-408Bassani, R.A., Bassani, J.W., Bers, D.M., Mitochondrial and sarcolemmal Ca2+ transport reduce [Ca2+]i during caffeine contractures in rabbit cardiac myocytes (1992) J Physiol, 453, pp. 591-608Voigt, N., Heijman, J., Wang, Q., Chiang, D.Y., Li, N., Karck, M., Cellular and molecular mechanisms of atrial arrhythmogenesis in patients with paroxysmal atrial fibrillation (2014) Circulation, 129, pp. 145-156Boyd, J.H., Mathur, S., Wang, Y., Bateman, R.M., Walley, K.R., Toll-like receptor stimulation in cardiomyoctes decreases contractility and initiates an NF-kappaB dependent inflammatory response (2006) Cardiovasc Res, 72, pp. 384-393Oyama, J., Blais, C., Liu, X., Pu, M., Kobzik, L., Kelly, R.A., Reduced myocardial ischemia-reperfusion injury in toll-like receptor 4-deficient mice (2004) Circulation, 109, pp. 784-789Zheng, Z.J., Croft, J.B., Giles, W.H., Mensah, G.A., Sudden cardiac death in the United States, 1989 to 1998 (2001) Circulation, 104, pp. 2158-2163Friedman, R.A., Kearney, D.L., Moak, J.P., Fenrich, A.L., Perry, J.C., Persistence of ventricular arrhythmia after resolution of occult myocarditis in children and young adults (1994) J Am Coll Cardiol, 24, pp. 780-783Wondergem, R., Graves, B.M., Ozment-Skelton, T.R., Li, C., Williams, D.L., Lipopolysaccharides directly decrease Ca2+ oscillations and the hyperpolarization-activated nonselective cation current If in immortalized HL-1 cardiomyocytes (2010) Am J Physiol Cell Physiol, 299, pp. C665-C671Medei, E., Marocolo, M., Rodrigues, D., Arantes, P.C., Takiya, C.M., Silva, J., Chronic treatment with anabolic steroids induces ventricular repolarization disturbances: cellular, ionic and molecular mechanism (2010) J Mol Cell Cardiol, 49, pp. 165-175Zhou, C., Ziegler, C., Birder, L.A., Stewart, A.F., Levitan, E.S., Angiotensin II and stretch activate NADPH oxidase to destabilize cardiac Kv4.3 channel mRNA (2006) Circ Res, 98, pp. 1040-1047Zhang, G., Ghosh, S., Toll-like receptor-mediated NF-kappaB activation: a phylogenetically conserved paradigm in innate immunity (2001) J Clin Invest, 107, pp. 13-19Takeda, K., Akira, S., TLR 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Le aziende turistiche "in rete"

Le aziende turistiche si trovano ad affrontare un’arena sem-pre più competitiva in un mercato che potenzialmente si al-larga sino a comprendere i confini dell’intero globo. Tra le risposte che vengono date dalle aziende, soprattutto quelle di piccole e medie dimensioni, in questo libro si delineano le diverse dimensioni dell’inserirsi in una “rete”. La rete di imprese si origina quando nell’azienda si definisce l’esigenza di creare accordi cooperativi con le altre azien-de . Tale concetto non rappresenta una novità nell’ambito eco-nomico aziendale e della ragioneria e rientra nella più ampia e vasta progenie delle aggregazioni aziendali, come al-cuni precursori sottolineano: “per questi motivi, la ragione-ria, che è la disciplina della interpretazione e rilevazione quantitativa dei fenomeni aziendali, deve inserire nei suoi studi teorici e pratici, l’indagine sulle relazioni interaziendali in quella forma ed ampiezza che saranno successivamente chiarite. In altri termini la ragioneria, fornendo dati a valore e dati quantitativi opportunamente elaborati ed interpretati, deve contribuire alla migliore conoscenza delle correlazioni ed interdipendenze fra gli organismi aziendali” . Sono stati diversi gli studiosi che hanno affrontato tale ar-gomento attribuendo alla rete le dimensioni economico-quantitative e contemporaneamente quelle di carattere quali-tativo-relazionali . Nelle aziende turistiche si è delineato an-che il concetto di “turismo relazionale”proprio per sottoli-neare le dinamiche qualitative degli accordi e delle relazio-ni che si sviluppano con essi . All’attuale stato dell’arte, però occorre ritornare a riflettere sul concetto di rete, anche in una prospettiva transdisciplina-re, perché la definizione che è stata proposta in passato dai vari studiosi, si arricchisce di ulteriori dimensioni, tra cui: la rete virtuale; il contratto di rete, la sostenibilità e l’accessibilità. Questo libro ha l’obiettivo di riflettere sugli aspetti appena menzionati, come prima fase di una riflessione, che è tuttora in corso di svolgimento e che richiede lo scorrere del tempo per cogliere al meglio i fenomeni analizzati. Il lavoro si suddivide in due parti principali, di cui la prima riguarda la riflessione che si basa su casi di ricerca e sulle diverse modalità che le aziende stanno sviluppando nelle “pieghe” delle reti sia virtuali, sia operative. La seconda parte presenta casi empirici, che suscitano ulte-riori riflessioni nel carattere fluido che il concetto di “rete” assume in questo periodo contingente. Siamo infatti in una fase, che possiamo definire “transito-ria”, dove la rete virtuale sta entrando nell’amministrazione aziendale ma trova a volte ostacoli nella cultura delle azien-de, che perciò si sentono a volte disorientate nell’apparente e, forse, fondato paradosso del rapporto tra la carenza di beni relazionali, che viene sottolineata da insigni studiosi e la rete virtuale , che tende sempre di più da un lato ad essere indispensabile per la sopravvivenza dell’azienda, come ab-biamo evidenziato di seguito ma dall’altro riduce ( o almeno sembra) la relazione facendola più superficiale e meno au-tentica. Su questo fronte si affacciano altre variabili nell’amministrazione delle aziende turistiche, in particolare quelle legate alla responsabilità sociale, alla sostenibilità e all’accessibilità, che sembrano trovano difficoltà a “connet-tersi” con altre dimensioni, quelle economiche e finanziarie, che, sino all’attuale stato dell’arte, sono state le uniche ad essere considerate importanti. In questa dinamica la riflessione non può non includere an-che approcci che provengono da altre discipline ed anche le professioni contabili, che da secoli camminano al fianco delle aziende turistiche cercando di prestare il loro fonda-mentale servizio. Al termine di questa introduzione vorrei ringraziare di cuore gli autori, che hanno contribuito alla realizzazione di questo lavoro e che continuano ad interrogarsi sulle evoluzioni future della “rete

Community-based Enterprises in Italy: Definition and Governance Models

In recent years, a new form of co-operative business is taking shape in Italy: the community-based enterprise, a community acting collectively at both management and business level, to pursue common goals. Community-based enterprises grow on a system of networks of free relationships among the members of a local community, with a high degree of reciprocity; when individuals work together in a business systems, relationships become social relationships. The complex of the community members’ relationships, objectives and expectations towards the company reflects necessarily on governance models that should follow a multistakeholder pattern. Multistakeholdership implies a direct and active participation of subjects bearing conflicting interests in the decisional process. This paper describes community-based enterprises in Italy, draws their governance models, and emphasizes how the passage from opening to different stakeholders to implementation of multistakeholder governance is very slow and difficult to put into practice