Eva M. Graf

Electrophysiological properties of human mesenchymal stem cells

Human mesenchymal stem cells (hMSC) have gained considerable interest due to their potential use for cell replacement therapy and tissue engineering. One strategy is to differentiate these bone marrow stem cells in vitro into cardiomyocytes prior to implantation. In this context ion channels can be important functional markers of cardiac differentiation. At present there is little information about the electrophysiological behaviour of the undifferentiated hMSC. We therefore investigated mRNA expression of 26 ion channel subunits using semiquantitative RT‐PCR and recorded transmembrane ion currents with the whole‐cell voltage clamp technique. Bone marrow hMSC were obtained from healthy donors. The cells revealed a distinct pattern of ion channel mRNA with high expression levels for some channel subunits (e.g. Kv4.2, Kv4.3, MaxiK, HCN2, and α1C of the L‐type calcium channel). Outward currents were recorded in almost all cells. The most abundant outward current rapidly activated at potentials positive to +20 mV. This current was identified as a large‐conductance voltage‐ and Ca2+‐activated K+ current, conducted by MaxiK channels, due to its high sensitivity to tetraethylammonium (IC50= 340 μm) and its inhibition by 100 nm iberiotoxin. A large fraction of cells also demonstrated a more slowly activating current at potentials positive to –30 mV. This current was selectively inhibited by clofilium (IC50= 0.8 μm). Ba2+ inward currents, stimulated by 1 μm BayK 8644 were found in a few cells, indicating the expression of functional L‐type Ca2+ channels. Other inward currents such as sodium currents or inward rectifier currents were absent. We conclude that undifferentiated hMSC express a distinct pattern of ion channel mRNA and functional ion channels that might contribute to physiological cell function.

Expression and function of dipeptidyl-aminopeptidase-like protein 6 as a putative β-subunit of human cardiac transient outward current encoded by Kv4.3

Dipeptidyl‐aminopeptidase‐like protein 6 (DPPX) was recently shown in the brain to modulate the kinetics of transient A‐type currents by accelerating inactivation and recovery from inactivation. Since the kinetics of human cardiac transient outward current (Ito) are not mimicked by coexpression of the α‐subunit Kv4.3 with its known β‐subunit KChIP2, we have tested the hypothesis that DPPX may serve as an additional β‐subunit in the human heart. With quantitative real‐time RT‐PCR strong mRNA expression of DPPX was detected in human ventricles and was verified at the protein level in human but not in rat heart by a DPPX‐specific antibody. Co‐expression of DPPX with Kv4.3 in Chinese hamster ovary cells produced Ito‐like currents, but compared with expression of KChIP2a and Kv4.3, the time constant of inactivation was faster, the potential of half‐maximum steady‐state inactivation was more negative and recovery from inactivation was delayed. Co‐expression of DPPX in addition to Kv4.3 and KChIP2a produced similar current kinetics as in human ventricular myocytes. We therefore propose that DPPX is an essential component of the native cardiac Ito channel complex in human heart.

5-Azacytidine induces changes in electrophysiological properties of human mesenchymal stem cells.

Previously, mouse bone marrow-derived stem cells (MSC) treated with the unspecific DNA methyltransferase inhibitor 5-azacytidine were reported to differentiate into cardiomyocytes. The aim of the present study was to investigate the efficiency of a similar differentiation strategy in human mononuclear cells obtained from healthy bone marrow donors. After 1–3 passages, cultures were exposed for 24 h to 5-azacytidine (3 μM) followed by 6 weeks of further culture. Drug treatment did not induce expression of myogenic marker MyoD or cardiac markers Nkx2.5 and GATA-4 and did not yield beating cells during follow-up. In patch clamp experiments, approximately 10-15% of treated and untreated cells exhibited L-type Ca2+ currents. Almost all cells showed outwardly rectifying K+ currents of rapid or slow activation kinetics. Mean current amplitude at +60 mV doubled after 6 weeks of treatment compared with time-matched controls. Membrane capacitance of treated cells was significantly larger than in controls 2 weeks after treatment and remained high after 6 weeks. Expression levels of mRNAs for the K+ channels Kv1.1, Kv1.5, Kv2.1, Kv4.3 and KCNMA1 and for the Ca2+ channel Cav1.2 were not affected by 5-azacytidine. Treatment with potassium channel blockers tetraethylammonium and clofilium at concentrations shown previously to inhibit rapid or slowly activating K+ currents of hMSC inhibited proliferation of these cells. Our results suggest that despite the absence of differentiation of hMSC into cardiomyocytes, treatment with 5-azacytidine caused profound changes in current density.

Molecular and Functional Expression of Voltage-Operated Calcium Channels During Osteogenic Differentiation of Human Mesenchymal Stem Cells

We used the patch‐clamp technique and RT‐PCR to study the molecular and functional expression of VOCCs in undifferentiated hMSCs and in cells undergoing osteogenic differentiation. L‐type Ca2+ channel blocker nifedipine did not influence alkaline phosphatase activity, calcium, and phosphate accumulation of hMSCs during osteogenic differentiation. This study suggests that osteogenic differentiation of hMSCs does not require L‐type Ca2+ channel function.

Identification and characterization of G beta 3s2, a novel splice variant of the G-protein beta 3 subunit.

The T-allele of a polymorphism (C825T) in the gene for the G-protein beta 3 subunit (GNB3) is associated with cardiovascular and metabolic disorders, distinct cellular features and altered drug responses. The molecular mechanisms that give rise to this complex phenotype have been linked to the occurrence of G beta 3s, a splice variant of GNB3. G beta 3s is predominantly expressed in cells with the 825T-allele. In the present study we describe the identification and characterization of an additional G beta 3 splice variant referred to as G beta 3s2. Its mRNA is expressed in heart, blood cells and tumour tissue, and its expression is also tightly associated with the GNB3 825T-allele. G beta 3s2 is generated by alternative splicing using non-canonical splice sites. G beta subunits belong to the family of propeller proteins and consist of seven regular propeller blades. Transcripts for G beta 3s2 are lacking 129 bp of the coding sequence of the wild-type G beta 3 protein. Thus the predicted structure consists of only six propeller blades, which resembles the structure of G beta 3s. Co-immunoprecipitation analyses indicated that G beta 3s2 dimerizes with different G gamma subunits, e.g. G gamma 5, G gamma 8(C) and G gamma 12. In Sf9 insect cells, expression of G beta 3s2 together with G gamma 12 enhances receptor-stimulated activation of G alpha(i2). Expression of G beta 3s2 in mammalian cells activated the mitogen-activated protein kinase cascade. Together, these results suggest that G beta 3s2 is a biologically active G beta variant which may play a role in the manifestation of the complex phenotype associated with the 825T-allele.

Differentiation induction of mouse embryonic stem cells into sinus node-like cells by suramin

BACKGROUND Embryonic stem (ES) cells differentiate into cardiac phenotypes representing early pacemaker-, atrial-, ventricular-, and sinus node-like cells, however, ES-derived specification into sinus nodal cells is not yet known. By using the naphthylamine derivative of urea, suramin, we were able to follow the process of cardiac specialization into sinus node-like cells. METHODS Differentiating mouse ES cells were treated with suramin (500 µM) from day 5 to 7 of embryoid body formation, and cells were analysed for their differentiation potential via morphological analysis, flow cytometry, RT-PCR, immunohistochemistry and patch clamp analysis. RESULTS Application of suramin resulted in an increased number of cardiac cells, but inhibition of neuronal, skeletal muscle and definitive endoderm differentiation. Immediately after suramin treatment, a decreased mesendoderm differentiation was found. Brachyury, FGF10, Wnt8 and Wnt3a transcript levels were significantly down-regulated, followed by a decrease in mesoderm- and cardiac progenitor-specific markers BMP2, GATA4/5, Wnt11, Isl1, Nkx2.5 and Tbx5 immediately after removal of the substance. With continued differentiation, a significant up-regulation of Brachyury, FGF10 and GATA5 transcript levels was observed, whereas Nkx2.5, Isl1, Tbx5, BMP2 and Wnt11 levels were normalized to control levels. At advanced differentiation stages, sinus node-specific HCN4, Tbx2 and Tbx3 transcript levels were significantly up-regulated. Immunofluorescence and patch-clamp analysis confirmed the increased number of sinus node-like cells, and electrophysiological analysis revealed a lower number of atrial- and ventricular-like cardiomyocytes following suramin treatment. CONCLUSION We conclude that the interference of suramin with the cardiac differentiation process modified mesoderm- and cardiac-specific gene expression resulting in enhanced formation of sinus node-like cells.

Murine ventricular L-type Ca(2+) current is enhanced by zinterol via beta(1)-adrenoceptors, and is reduced in TG4 mice overexpressing the human beta(2)-adrenoceptor.

The functional coupling of β2‐adrenoceptors (β2‐ARs) to murine L‐type Ca2+ current (ICa(L)) was investigated with two different approaches. The β2‐AR signalling cascade was activated either with the β2‐AR selective agonist zinterol (myocytes from wild‐type mice), or by spontaneously active, unoccupied β2‐ARs (myocytes from TG4 mice with 435 fold overexpression of human β2‐ARs). Ca2+ and Ba2+ currents were recorded in the whole‐cell and cell‐attached configuration of the patch‐clamp technique, respectively. Zinterol (10 μM) significantly increased ICa(L) amplitude of wild‐type myocytes by 19±5%, and this effect was markedly enhanced after inactivation of Gi‐proteins with pertussis‐toxin (PTX; 76±13% increase). However, the effect of zinterol was entirely mediated by the β1‐AR subtype, since it was blocked by the β1‐AR selective antagonist CGP 20712A (300 nM). The β2‐AR selective antagonist ICI 118,551 (50 nM) did not affect the response of ICa(L) to zinterol. In myocytes with β2‐AR overexpression ICa(L) was not stimulated by the activated signalling cascade. On the contrary, ICa(L) was lower in TG4 myocytes and a significant reduction of single‐channel activity was identified as a reason for the lower whole‐cell ICa(L). The β2‐AR inverse agonist ICI 118,551 did not further decrease ICa(L). PTX‐treatment increased current amplitude to values found in control myocytes. In conclusion, there is no evidence for β2‐AR mediated increases of ICa(L) in wild‐type mouse ventricular myocytes. Inactivation of Gi‐proteins does not unmask β2‐AR responses to zinterol, but augments β1‐AR mediated increases of ICa(L). In the mouse model of β2‐AR overexpression ICa(L) is reduced due to tonic activation of Gi‐proteins.

Changes in morphology and inward rectifier currents in human atrial myocytes depend on culture conditions

Abstract. Human atrial myocytes were cultured under systematically varied conditions in order to obtain stable cells for future gene manipulation. Transient (Ito) and sustained outward current (Iso), and voltage- and muscarinic receptor-activated inward rectifier K+ currents (IK1, IK,ACh) were measured in freshly isolated cells and after 5 days in culture. Myocytes were grown on polylysin or laminin in medium with or without 10 % serum (medium+S, medium-S). Cultured myocytes dedifferentiated to a greater extent in medium+S than medium-S, but independent of the chemical nature of the adherence surface. Apparent surface area increased in medium+S, whereas membrane capacitance declined under all culture conditions. Ito of myocytes cultured in medium-S was increased. Myocytes grown on polylysin and laminin exhibited reduced IK1 current density. Under all culture conditions, IK,ACh was attenuated with carbachol but hardly affected with sphingosine-1-phosphate as agonists. In conclusion, morphological and electrophysiological changes depended on serum in the culture medium rather than on adherence surface being coated with laminin or polylysin.

Genes and stem cells: New therapeutical concepts

ZusammenfassungDie moderne Behandlung des akuten Myokardinfarkts und anderer Ischämie bedingter Herzerkrankungen hat zwar deutliche Verbesserungen hinsichtlich der akuten Sterblichkeit gebracht, dennoch bleibt die schwer aufzuhaltende Verschlechterung der Myokardfunktion im Langzeitverlauf ein bisher nicht befriedigend gelöstes therapeutisches Problem. In jüngster Zeit haben neue Therapieansätze wie Implantation von Stammzellen oder Gentherapie ein breites Interesse gefunden. Diese Strategien zielen auf den Ersatz von zugrundegegangenen Herzmuskelzellen und die Revaskularisierung des gefährdeten Myokardbereichs ab, um das zerstörte Gewebe zu erneuern. Einige dieser Konzepte werden bereits klinisch erprobt und haben zu ermutigenden Ergebnissen geführt. In der vorliegenden Arbeit werden die Eigenschaften von Stammzellen beschrieben, geeignete Quellen für die Gewinnung einer ausreichenden Zellzahl diskutiert und über Ergebnisse aus sowohl experimentellem als auch klinischem Einsatz zur Regeneration des Myokards berichtet. Unter den therapeutischen Strategien scheinen die Anwendung von mesenchymalen Stammzellen aus dem Knochenmark und die Gentherapie mit vaskulären Wachstumsfaktoren die am weitesten entwickelten Methoden mit der größten Aussicht auf therapeutischen Erfolg zu sein. Dennoch ist es dringend notwendig, unser Verständnis von biologischen Regenerationsvorgängen zu vertiefen, um die neuartigen Therapieansätze zu verbessern.SummaryModern treatment of acute myocardial infarction and other ischemia-related heart diseases has enhanced acute survival, but the long-term problem of deterioration of myocardial function due to loss of damaged myocytes remains a therapeutic challenge. In this context, new approaches including stem cell implantation and gene therapy have gained much scientific interest. These strategies aim at replacement of lost cardiomyocytes and at revascularization of tissue areas at risk in order to regenerate destroyed myocardium. Though still in an experimental stage, some of these concepts are currently being tested in patients with encouraging results. Here we will describe stem cell properties, suitable sources that could provide sufficient cell numbers and experimental as well as clinical results of their application for regeneration of the myocardium. Among the therapeutic strategies, bone marrow-derived stem cells and gene therapy with vascular growth factors are presented as the most promising and advanced methods presently available. Nevertheless, we require still better insights into biology of regeneration processes in order to improve these novel therapeutic procedures.