Transfektionsoptimierung an Primärkulturen hippokampaler Neurone

In mehreren Versuchsreihen soll unter schrittweisem Verändern einzelner Parameter die Transfektionseffizienz an hippokampalen Neuronen optimiert werden. Es werden die Transfektionsreagenzien CalPhosTM, LipofectamineTM 2000, GenCarrier-2TM und FuGENE® HD (verbreitete, kommerziell erhältliche Transfektionsreagenzien) anhand der Transfektion von GFP miteinander verglichen. Anhand von elektrophysiologischen Messungen wird der Zustand der transfizierten Zellen beurteilt. Der Vergleich der Transfektionsreagenzien zeigte, dass nur mit CalPhosTM und LipofectamineTM 2000 ein verwendbarer Transfektionserfolg zu erzielen war. Außerdem war eine starke Abhängigkeit des Transfektionserfolges vom Alter der Neurone zu erkennen. Neurone die unter 7 Tage alt waren, erzielten die höchsten Transfektionserfolge. Bei der Variation der LipofectamineTM 2000 Menge zeigte sich, dass niedrigere Konzentrationen einen wesentlich höheren Erfolg lieferten als höhere Konzentrationen LipofectamineTM 2000 und dass höhere Konzentrationen die Zellen zunehmend schädigten, was auch durch die elektrophysiologischen Messungen sichtbar wurde. Anhand zweier unterschiedlicher LipofectamineTM 2000 Konzentrationen wurden die Auswirkungen verschiedener DNA Mengen auf die Transfektionseffizienz getestet. Es wurde ein weitaus höherer Transfektionserfolg mit DNA Mengen über 1 μg als mit Mengen unter 1 μg DNA sichtbar. Als letzter Parameter wurde der Einfluss von Medium-Zusatzstoffen wie Serum Supplements (Nuts) und einem Hemmer von Glutamat-Rezeptor-abhängiger Erregungstoxizität (KYN) getestet. Diese Stoffe zeigten keinen Einfluss auf den Transfektionserfolg. Mit einer LipofectamineTM 2000 Menge von 7,5 μL und einer DNA Menge von über 1 μg konnte ein, mit einer Effizienz von über 5 %, zumindest für elektrophysiologische Versuche ansprechender Transfektionserfolg erzielt werden.In this diploma thesis the transfection reagents CalPhosTM, LipofectamineTM 2000, GenCarrier-2TM and FuGENE®HD, widely used, commercially available transfection reagents, have been compared and tested on primary hippocampal neurons. To optimize the transfection efficiency, different parameters were varied. Additionally the condition of transfected cells was evaluated by electrophysiological measurements. The comparison of the different transfection reagents indicated, that only CalPhosTM and LipofectamineTM 2000 led to an acceptable transfection success. Furthermore, a dependence of transfection efficiency on the cell-age was observed. Experiments on cells of different ages showed that best transfection success was obtained with cells younger than 7 days. The optimal concentration of LipofectamineTM 2000 was tested in another series of experiments. An even better success with low concentration of LipofectamineTM 2000 and a destructive effect, when high concentrations of LipofectamineTM2000 were used, was observed. The effect on transfection efficiency with different DNA concentrations was tested by using 7,5μL and 15μL LipofectamineTM 2000. With both concentrations of LipofectamineTM2000 a better success of transfection was observed with DNA concentrations higher than 1μg, but further increasing the DNA amount did not continue to enhance transfection rates. In the last set of experiments the influence of medium-supplements, like Nuts and KYN, was investigated, but these additions did not reveal any positive effect on transfection rates. In conclusion I report, that for hippocampal neurons in primary culture, 7,5μL LipofectamineTM 2000 and more than 1μg DNA, when applied on young neurons ( 5%)

Voltage-gated ion channel impairments in dystrophic cardiomyocytes

Introduction/Background: The muscular dystrophies (MDs) are a group of inherited muscular disorders, which are characterized by progressive muscle weakening and wasting. Six different groups can be classified, among which are the dystrophinopathies (e.g. Duchenne muscular dystrophy, DMD) and the limb-girdle muscular dystrophies (e.g. LGMD2B). DMD is the most common and severe form among the MDs. Both DMD (severe) and LGMD2B (mild) are characterized by cardiac pathology and dysfunction. The cardiac phenotype may be related to impairments in the expression and function of voltage-gated ion channels. One of the most important ion channels in the heart is the L-type voltage-gated Ca2+ channel, which is involved in action potential shaping and in the inition of contraction. The main aim of this study was to identify potential abnormalities in L-type Ca2+ channel function in mouse models of DMD and LGMD2B. Methods and Results: To study ion channel abnormalities in the dystrophic heart, isolated single ventricular cardiomyocytes from adult DMD (mdx, dystrophin-deficient; mdx-utr, dystrophin- and utrophin-deficient) and LGMD2B (dysf, dysferlin deficient) mouse models were compared to wild type (wt) cells. In dystrophin-deficient cardiomyocytes currents through L-type Ca2+ channels were increased and channel inactivation was reduced. To study the functional effects of abnormal Ca2+ channels, action potentials (APs) of dystrophin-deficient cardiomyocytes and electrocardiograms (ECGs) from anaesthetised dystrophin-deficient mice were recorded. Although AP duration was unchanged, Ca2+ current dependent ECG parameters in dystrophin-deficient hearts were significantly altered. Western blot and immunofluorescence experiments showed no differences in Ca2+ channel protein levels or localization between dystrophin-deficient and wt cardiomyocytes. To identify the reasons for abnormal Ca2+ channel properties in dystrophin-deficient cardiomyocytes, electrophysiological measurements modulating protein kinase A (PKA) and neuronal nitric oxide synthase (nNOS) were performed. There was no difference in the response of Ca2+ currents to stimulation or inhibition of the PKA-mediated pathway in wt and mdx cardiomyocytes, but there was a difference in the channel's response to nNOS pathway modulation. Furthermore nNOS levels seemed to be reduced in mdx cardiomyocytes. Finally, there were no significant differences between wt and dysf cardiomyocytes regarding Ba2+, Ca2+ and Na+ currents and APs, as well as normal ECG parameters in dysferlin deficient mice. Conclusions: (1) Significant abnormalities of L-type Ca2+ channels in dystrophin-deficient cardiomyocytes, but no alterations in cardiac voltage-gated ion channels in dysferlin deficient cardiomyocytes suggest that dystrophin regulates cardiac ion channels, whereas dysferlin does not. Thus, abnormalities in voltage-gated ion channels do not represent a common feature of all types of MDs. (2) The enhanced Ca2+ currents in dystrophin-deficient cardiomyocytes may be caused by a reduced nNOS activity in these cells. (3) The observed gain of L-type Ca2+ channel function in dystrophin-deficient cardiomyocytes may disturb the electrophysiology of the dystrophic heart and thereby contribute to the cardiac pathology observed in DMD patients.Einleitung/Hintergrund: Muskeldystrophien bilden eine Gruppe vererbter Muskelkrankheiten, die durch den fortschreitenden Muskelabbau und eine Muskelschwächung charakterisiert sind. Es können 6 Klassen unterschieden werden, unter welchen sich auch die Dystrophinopathien (z.B.: Duchenne Muskeldystrophie, DMD) und die Gliedergürtel-Muskeldystrophien (z.B.: LGMD2B) befinden. DMD ist die am häufigsten auftretende und schwerwiegenste Form der Muskeldystrophien. Für beide der genannten Muskeldystrophien (DMD, schwer; und LGMD2B, leicht) sind das Auftreten von Pathologien und Dysfunktionen des Herzens charakteristisch. Das Auftreten dieser kardialen Dysfunktionen könnte mit Beeinträchtigungen der Expression oder der Funktion von spannungsgesteuerten Ionenkanälen in Beziehung stehen. Einer der wichtigsten Ionenkanäle des Herzens ist der spannungsgesteuerte Ca2+ Kanal des L-Types, der bei der Formgebung von Aktionspotentialen und der Initiierung der Muskelkontraktion eine Rolle spielt. Das Hauptziel dieser Studie war es, mögliche Abnormitäten der L-Typ Ca2+ Kanalfunktion in Mausmodellen für DMD und LGMD2B aufzudecken. Methoden und Ergebnisse: Um potentielle Abnormalitäten von Ionenkanälen im dystrophen Herzen zu studieren, wurden einzelne isolierte ventrikuläre Kardiomyozyten von adulten Mausmodellen für DMD (mdx, Dystrophin-defizient; und mdx-utr, Dystrophin- und Utrophin-defizient) und LGMD2B (dysf, Dysferlin-defizient) mit jenen von Wildtyp (wt)-Tieren verglichen. In Dystrophin-defizienten Kardiomyozyten fanden sich erhöhte L-Typ Ca2+ Kanalströme, sowie eine reduzierte Kanalinaktivierung. Um die funktionellen Effekte der abnormen Ca2+ Kanäle zu studieren, wurden Aktionspotentiale einzelner Dystrophin-defizienter Kardiomyozyten, sowie Elektrokardiogramme von narkotisierten Dytrophin-defizienten Mäusen gemessen. Obwohl die Aktionspotentialdauer unverändert war, zeigten Ca2+-abhängige Parameter der von Dsytrophin-defizienten Mäusen abgeleiteten Elektrokardiogramme signifikante Änderungen. Westernblot- und Immunfluoreszenzexperimente zeigten keine Änderungen des Proteinlevels oder der Lokalisierung des Ca2+ Kanalproteins in Dystrophin-defizienten Herzzellen. Um die Ursache der abnormen Kanaleigenschaften in Dystrophin-defizienten Kardiomyozyten zu ermitteln, wurden elektrophysiologische Messungen durchgeführt in welchen Proteinkinase A (PKA) und die neuronale Stickstoffmonoxid-Synthase (nNOS) moduliert wurden. Es gab keine Unterschiede in der Ca2+ Stromantwort auf die Stimulierung oder die Inhibierung des PKA-vermittelten Signalweges in wt und mdx Kardiomyozyten, jedoch gab es einen Unterschied in der Kanalantwort auf die Modulation des nNOS-vermittelten Signalweges zwischen den beiden Genotypen. Auch der nNOS Spiegel in mdx Kardiomyozyten schien erniedrigt. Es gab keine signifikanten, die Ba2+, Ca2+ und Na+ Ströme-, ebenso wie Aktionspotential- oder Elektrokardiogramm-betreffenden Unterschiede, zwischen wt und Dysferlin defizienten dysf Mäusen. Fazit: (1) Signifikante Abnormalitäten des L-Typ Ca2+ Kanals in Dystrophin-defizienten Kardiomyozyten, aber keine Änderungen der kardialen spannungsgesteuerten Ionenkanäle in Dysferlin-defizienten Herzzellen lassen vermuten, dass Dystrophin Ionenkanäle des Herzens reguliert, Dysferlin hingegen nicht. Daher kann geschlussfolgert werden, dass spannungsgesteuerte Ionenkanalabnormalitäten kein gemeinsames Merkmal aller Muskeldystrophiearten darstellt. (2) Die erhöhten Ca2+ Ströme in Dystrophin-defizienten Kardiomyozyten könnten durch eine reduzierte nNOS Aktivität in diesen Zellen verursacht sein. (3) Der beobachtete Anstieg der L-Typ Ca2+ Kanalfunktion in Dystrophin-defizienten Kardiomyozyten könnte die Elektrophysiologie des dystrophen Herzens beeinträchtigen und auf diese Art zu den, in DMD Patienten beobachteten, Herzpathologien beitragen.Abweichender Titel laut Übersetzung der Verfasserin/des VerfassersWien, Med. Univ., Diss., 2015(VLID)171456

Physiological Reports / Calcium current properties in dystrophindeficient ventricular cardiomyocytes from aged mdx mice

Duchenne muscular dystrophy (DMD), caused by mutations in the gene encoding for the cytoskeletal protein dystrophin, is linked with severe cardiac complications including cardiomyopathy development and cardiac arrhythmias. We and others recently reported that currents through Ltype calcium (Ca) channels were significantly increased, and channel inactivation was reduced in dystrophindeficient ventricular cardiomyocytes derived from the mdx mouse, the most commonly used animal model for human DMD. These gainoffunction Ca channel abnormalities may enhance the risk of Cadependent arrhythmias and cellular Ca overload in the dystrophic heart. All studies, which have so far investigated Ltype Ca channel properties in dystrophic cardiomyocytes, have used hearts from either neonatal or young adult mdx mice as cell source. In consequence, the dimension of the Ca channel abnormalities present in the severelydiseased aged dystrophic heart has remained unknown. Here, we have studied potential abnormalities in Ca currents and intracellular Ca transients in ventricular cardiomyocytes derived from aged dystrophic mdx mice. We found that both the Ltype and Ttype Ca current properties of mdx cardiomyocytes were similar to those of myocytes derived from aged wildtype mice. Accordingly, Ca release from the sarcoplasmic reticulum was normal in cardiomyocytes from aged mdx mice. This suggests that, irrespective of the presence of a pronounced cardiomyopathy in aged mdx mice, Ca currents and Ca release in dystrophic cardiomyocytes are normal. Finally, our data imply that dystrophin regulation of Ltype Ca channel function in the heart is lost during aging.(VLID)483964

Channels / Decreased inward rectifier potassium current IK in dystrophin-deficient ventricular cardiomyocytes

Kir2.x channels in ventricular cardiomyocytes (most prominently Kir2.1) account for the inward rectifier potassium current IK, which controls the resting membrane potential and the final phase of action potential repolarization. Recently it was hypothesized that the dystrophin-associated protein complex (DAPC) is important in the regulation of Kir2.x channels. To test this hypothesis, we investigated potential IK abnormalities in dystrophin-deficient ventricular cardiomyocytes derived from the hearts of Duchenne muscular dystrophy mouse models. We found that IK was substantially diminished in dystrophin-deficient cardiomyocytes when compared to wild type myocytes. This finding represents the first functional evidence for a significant role of the DAPC in the regulation of Kir2.x channels.(VLID)488580

Dopamine depletion induces neuron‐specific alterations of GABAergic transmission in the mouse striatum

Lack of dopamine (DA) in the striatum and the consequential dysregulation of thalamocortical circuits are major causes of motor impairments in Parkinson's disease. The striatum receives multiple cortical and subcortical afferents. Its role in movement control and motor skills learning is regulated by DA from the nigrostriatal pathway. In Parkinson's disease, DA loss affects striatal network activity and induces a functional imbalance of its output pathways, impairing thalamocortical function. Striatal projection neurons are GABAergic and form two functionally antagonistic pathways: the direct pathway, originating from DA receptor type 1-expressing medium spiny neurons (D1 R-MSN), and the indirect pathway, from D2 R-MSN. Here, we investigated whether DA depletion in mouse striatum also affects GABAergic function. We recorded GABAergic miniature IPSCs (mIPSC) and tonic inhibition from D1 R- and D2 R-MSN and used immunohistochemical labeling to study GABAA R function and subcellular distribution in DA-depleted and control mice. We observed slower decay kinetics and increased tonic inhibition in D1 R-MSN, while D2 R-MSN had increased mIPSC frequency after DA depletion. Perisomatic synapses containing the GABAA R subunits α1 or α2 were not affected, but there was a strong decrease in non-synaptic GABAA Rs containing these subunits, suggesting altered receptor trafficking. To broaden these findings, we also investigated GABAA Rs in GABAergic and cholinergic interneurons and found cell type-specific alterations in receptor distribution, likely reflecting changes in connectivity. Our results reveal that chronic DA depletion alters striatal GABAergic transmission, thereby affecting cellular and circuit activity. These alterations either result from pathological changes or represent a compensatory mechanism to counteract imbalance of output pathways

Weakly supervised inference of personalized heart meshes based on echocardiography videos

Echocardiography provides recordings of the heart chamber size and function and is a central tool for non-invasive diagnosis of heart diseases. It produces high-dimensional video data with substantial stochasticity in the measurements, which frequently prove difficult to interpret. To address this challenge, we propose an automated framework to enable the inference of a high resolution personalized 4D (3D plus time) surface mesh of the cardiac structures from 2D echocardiography video data. Inferring such shape models arises as a key step towards accurate personalized simulation that enables an automated assessment of the cardiac chamber morphology and function. The proposed method is trained using only unpaired echocardiography and heart mesh videos to find a mapping between these distinct visual domains in a self-supervised manner. The resulting model produces personalized 4D heart meshes, which exhibit a high correspondence with the input echocardiography videos. Furthermore, the 4D heart meshes enable the automatic extraction of echocardiographic variables, such as ejection fraction, myocardial muscle mass, and volumetric changes of chamber volumes over time with high temporal resolution.ISSN:1361-8415ISSN:1361-842

Weakly supervised inference of personalized heart meshes based on echocardiography videos

Echocardiography provides recordings of the heart chamber size and function and is a central tool for non-invasive diagnosis of heart diseases. It produces high-dimensional video data with substantial stochasticity in the measurements, which frequently prove difficult to interpret. To address this challenge, we propose an automated framework to enable the inference of a high resolution personalized 4D (3D plus time) surface mesh of the cardiac structures from 2D echocardiography video data. Inferring such shape models arises as a key step towards accurate personalized simulation that enables an automated assessment of the cardiac chamber morphology and function. The proposed method is trained using only unpaired echocardiography and heart mesh videos to find a mapping between these distinct visual domains in a self-supervised manner. The resulting model produces personalized 4D heart meshes, which exhibit a high correspondence with the input echocardiography videos. Furthermore, the 4D heart meshes enable the automatic extraction of echocardiographic variables, such as ejection fraction, myocardial muscle mass, and volumetric changes of chamber volumes over time with high temporal resolution