Phosphoprotein Phosphatase 1 but Not 2A Activity Modulates Coupled-Clock Mechanisms to Impact on Intrinsic Automaticity of Sinoatrial Nodal Pacemaker Cells.

Spontaneous AP (action potential) firing of sinoatrial nodal cells (SANC) is critically dependent on protein kinase A (PKA) and Ca2+/calmodulin-dependent protein kinase II (CaMKII)-dependent protein phosphorylation, which are required for the generation of spontaneous, diastolic local Ca2+ releases (LCRs). Although phosphoprotein phosphatases (PP) regulate protein phosphorylation, the expression level of PPs and phosphatase inhibitors in SANC and the impact of phosphatase inhibition on the spontaneous LCRs and other players of the oscillatory coupled-clock system is unknown. Here, we show that rabbit SANC express both PP1, PP2A, and endogenous PP inhibitors I-1 (PPI-1), dopamine and cyclic adenosine 3',5'-monophosphate (cAMP)-regulated phosphoprotein (DARPP-32), kinase C-enhanced PP1 inhibitor (KEPI). Application of Calyculin A, (CyA), a PPs inhibitor, to intact, freshly isolated single SANC: (1) significantly increased phospholamban (PLB) phosphorylation (by 2-3-fold) at both CaMKII-dependent Thr17 and PKA-dependent Ser16 sites, in a time and concentration dependent manner; (2) increased ryanodine receptor (RyR) phosphorylation at the Ser2809 site; (3) substantially increased sarcoplasmic reticulum (SR) Ca2+ load; (4) augmented L-type Ca2+ current amplitude; (5) augmented LCR's characteristics and decreased LCR period in intact and permeabilized SANC, and (6) increased the spontaneous basal AP firing rate. In contrast, the selective PP2A inhibitor okadaic acid (100 nmol/L) had no significant effect on spontaneous AP firing, LCR parameters, or PLB phosphorylation. Application of purified PP1 to permeabilized SANC suppressed LCR, whereas purified PP2A had no effect on LCR characteristics. Our numerical model simulations demonstrated that PP inhibition increases AP firing rate via a coupled-clock mechanism, including respective increases in the SR Ca2+ pumping rate, L-type Ca2+ current, and Na+/Ca2+-exchanger current. Thus, PP1 and its endogenous inhibitors modulate the basal spontaneous firing rate of cardiac pacemaker cells by suppressing SR Ca2+ cycling protein phosphorylation, the SR Ca2+ load and LCRs, and L-type Ca2+ current.

Ca2+ and Membrane Potential Transitions During Action Potentials Are Self-Similar to Each Other and to Variability of AP Firing Intervals Across the Broad Physiologic Range of AP Intervals During Autonomic Receptor Stimulation.

Ca2+ and V m transitions occurring throughout action potential (AP) cycles in sinoatrial nodal (SAN) cells are cues that (1) not only regulate activation states of molecules operating within criticality (Ca2+ domain) and limit-cycle (V m domain) mechanisms of a coupled-clock system that underlies SAN cell automaticity, (2) but are also regulated by the activation states of the clock molecules they regulate. In other terms, these cues are both causes and effects of clock molecular activation (recursion). Recently, we demonstrated that Ca2+ and V m transitions during AP cycles in single SAN cells isolated from mice, guinea pigs, rabbits, and humans are self-similar (obey a power law) and are also self-similar to trans-species AP firing intervals (APFIs) of these cells in vitro, to heart rate in vivo, and to body mass. Neurotransmitter stimulation of ß-adrenergic receptor or cholinergic receptor-initiated signaling in SAN cells modulates their AP firing rate and rhythm by impacting on the degree to which SAN clocks couple to each other, creating the broad physiologic range of SAN cell mean APFIs and firing interval variabilities. Here we show that Ca2+ and V m domain kinetic transitions (time to AP ignition in diastole and 90% AP recovery) occurring within given AP, the mean APFIs, and APFI variabilities within the time series of APs in 230 individual SAN cells are self-similar (obey power laws). In other terms, these long-range correlations inform on self-similar distributions of order among SAN cells across the entire broad physiologic range of SAN APFIs, regardless of whether autonomic receptors of these cells are stimulated or not and regardless of the type (adrenergic or cholinergic) of autonomic receptor stimulation. These long-range correlations among distributions of Ca2+ and V m kinetic functions that regulate SAN cell clock coupling during each AP cycle in different individual, isolated SAN cells not in contact with each other. Our numerical model simulations further extended our perspectives to the molecular scale and demonstrated that many ion currents also behave self-similar across autonomic states. Thus, to ensure rapid flexibility of AP firing rates in response to different types and degrees of autonomic input, nature "did not reinvent molecular wheels within the coupled-clock system of pacemaker cells," but differentially engaged or scaled the kinetics of gears that regulate the rate and rhythm at which the "wheels spin" in a given autonomic input context.

Polar aromatic periphery increases agonist potency of spirocyclic free fatty acid receptor (GPR40) agonists inspired by LY2881835.

A series of spirocyclic compounds inspired by Eli Lilly's phase 1 antidiabetic FFA1 receptor agonist LY2881835 was designed to include polar aromatic periphery groups and explore a possibility of building additional contacts with the target near the agonist binding site. The frontrunner compound in the series (9i) was shown to be a potent (EC50 = 260 nM) FFA1 agonist with excellent aqueous (PBS) solubility and good Caco-2 permeability. The observed structure-activity relationships were rationalized by a docking study. The new series significantly expands the ligand landscape for the ongoing quest for new potent and more polar FFA1 agonists as fundamentally new class of therapeutic agents against type 2 diabetes mellitus.

Novel FFA1 (GPR40) agonists containing spirocyclic periphery: polar azine periphery as a driver of potency.

A series of nine compounds based on 3-[4-(benzyloxy)phenyl]propanoic acid core containing a 1-oxa-9-azaspiro[5.5]undecane periphery was designed, synthesized and evaluated as free fatty acid 1 (FFA1 or GPR40) agonists. The spirocyclic appendages included in these compounds were inspired by LY2881835, Eli Lilly's advanced drug candidate for type II diabetes mellitus that was in phase I clinical trials. These polar spirocyclic, fully saturated appendages (that are themselves uncharacteristic of the known FFA1 ligand space) were further decorated with diverse polar groups (such as basic heterocycles or secondary amides). To our surprise, while seven of nine compounds were found to be inactive (likely due to the decrease in lipophilicity, which is known to be detrimental to FFA1 ligand affinity), two compounds containing 2-pyridyloxy and 2-pyrimidinyloxy groups were found to have EC50 of 1.621 and 0.904 µM, respectively. This result is significant in the context of the worldwide quest for more polar FFA1 agonists, which would be devoid of liver toxicity effects earlier observed for a FFA1 agonist fasiglifam (TAk-875) in clinical studies.

Free fatty acid receptor 1 (GPR40) agonists containing spirocyclic periphery inspired by LY2881835.

The free fatty acid receptor 1 (FFA1), a G protein-coupled receptor (GPCR) naturally activated by long-chain fatty acids is a novel target for the treatment of metabolic diseases. The basic amine spirocyclic periphery of Eli Lilly's drug candidate LY2881835 for treatment of type 2 diabetes mellitus (which reached phase I clinical trials) inspired a series of novel FFA1 agonists. These were designed to incorporate the 3-[4-(benzyloxy)phenyl]propanoic acid pharmacophore core decorated with a range of spirocyclic motifs. The latter were prepared via the Prins cyclization and subsequent modification of the 4-hydroxytetrahydropyran moiety in the Prins product. Here, we synthesize 19 compounds and test for FFA1 activity. Within this pilot set, a nanomolar potency (EC50=55nM) was reached. Four lead compounds (EC50 range 55-410nM) were characterized for aqueous solubility, metabolic stability, plasma protein binding and Caco-2 permeability. While some instability in the presence of mouse liver microsomes was noted, mouse pharmacokinetic profile of the compound having the best overall ADME properties was evaluated to reveal acceptable bioavailability (F=10.3%) and plasma levels achieved on oral administration.

Novel free fatty acid receptor 1 (GPR40) agonists based on 1,3,4-thiadiazole-2-carboxamide scaffold.

Free fatty acid receptor 1 (FFA1), previously known as GPR40 is a G protein-coupled receptor and a new target for treatment of type 2 diabetes. Two series of FFA1 agonists utilizing a 1,3,4-thiadiazole-2-caboxamide scaffold were synthetized. Both series offered significant improvement of the potency compared to the previously described 1,3,4-thiadiazole-based FFA1 agonists and high selectivity for FFA1. Molecular docking predicts new aromatic interactions with the receptor that improve agonist potency. The most potent compounds from both series were profiled for in vitro ADME properties (plasma and metabolic stability, LogD, plasma protein binding, hERG binding and CYP inhibition). One series suffered very rapid degradation in plasma and in presence of mouse liver microsomes. However, the other series delivered a lead compound that displayed a reasonable ADME profile together with the improved FFA1 potency.

Novel agonists of free fatty acid receptor 1 (GPR40) based on 3-(1,3,4-thiadiazol-2-yl)propanoic acid scaffold.

1,3,4-Thiadiazole was explored as a more polar, heterocyclic replacement for the phenyl ring in the 3-arylpropionic acid pharmacophore present in the majority of GPR40 agonists. Out of 13 compounds synthesized using a flexible, three-step protocol (involving no chromatographic purification), four compounds were confirmed to activate the target in micromolar concentration range. While the potency of the series should be subject of further optimization, the remarkable aqueous solubility and microsomal stability observed for the lead compound (8g) apparently attests to this new scaffold's high promise in the GPR40 agonist field.

Phenoxymethyl 1,3-oxazoles and 1,2,4-oxadiazoles as potent and selective agonists of free fatty acid receptor 1 (GPR40).

A screening hit that showed a weak (EC50 = 18 µM), partial agonistic effect on GPR40 was used a prototype for expedited hit expansion effort using a set of advanced building blocks. The latter yielded several 1,3-oxazoles and 1,2,4-oxadiazoles with significantly improved potency (best EC50 = 0.058 µM). The lead compounds in each chemotype showed a very good ADME profile (aqueous solubility, plasma protein binding, microsomal stability and membrane permeability) and no appreciable inhibition of key cytochromes P450. The compounds reported are significant new starting points for further preclinical development of future diabetic agents with a mechanism of action for which a first-in-class agent is yet to be approved.

Rhythmic beating of stem cell-derived cardiac cells requires dynamic coupling of electrophysiology and Ca cycling.

There is an intense interest in differentiating embryonic stem cells to engineer biological pacemakers as an alternative to electronic pacemakers for patients with cardiac pacemaker function deficiency. Embryonic stem cell-derived cardiocytes (ESCs), however, often exhibit dysrhythmic excitations. Using Ca(2+) imaging and patch-clamp techniques, we studied requirements for generation of spontaneous rhythmic action potentials (APs) in late-stage mouse ESCs. Sarcoplasmic reticulum (SR) of ESCs generates spontaneous, rhythmic, wavelet-like Local Ca(2+)Releases (LCRs) (inhibited by ryanodine, tetracaine, or thapsigargin). L-type Ca(2+)current (I(CaL)) induces a global Ca(2+) release (CICR), depleting the Ca(2+) content SR which resets the phases of LCR oscillators. Following a delay, SR then generates a highly synchronized spontaneous Ca(2+)release of multiple LCRs throughout the cell. The LCRs generate an inward Na(+)/Ca(2+)exchanger (NCX) current (absent in Na(+)-free solution) that ignites the next AP. Interfering with SR Ca(2+) cycling (ryanodine, caffeine, thapsigargin, cyclopiazonic acid, BAPTA-AM), NCX (Na(+)-free solution), or I(CaL) (nifedipine) results in dysrhythmic excitations or cessation of automaticity. Inhibition of cAMP/PKA signaling by a specific PKA inhibitor, PKI, decreases SR Ca(2+) loading, substantially reducing both spontaneous LCRs (number, size, and amplitude) and rhythmic AP firing. In contrast, enhancing PKA signaling by cAMP increases the LCRs (number, size, duration) and converts irregularly beating ESCs to rhythmic "pacemaker-like" cells. SR Ca(2+) loading and LCR activity could be also increased with a selective activation of SR Ca(2+) pumping by a phospholamban antibody. We conclude that SR Ca(2+) loading and spontaneous rhythmic LCRs are driven by inherent cAMP/PKA activity. I(CaL) synchronizes multiple LCR oscillators resulting in strong, partially synchronized diastolic Ca(2+) release and NCX current. Rhythmic ESC automaticity can be achieved by boosting "coupling" factors, such as cAMP/PKA signaling, that enhance interactions between SR and sarcolemma.

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.

Cholinergic receptor signaling modulates spontaneous firing of sinoatrial nodal cells via integrated effects on PKA-dependent Ca(2+) cycling and I(KACh).

Prior studies indicate that cholinergic receptor (ChR) activation is linked to beating rate reduction (BRR) in sinoatrial nodal cells (SANC) via 1) a G(i)-coupled reduction in adenylyl cyclase (AC) activity, leading to a reduction of cAMP or protein kinase A (PKA) modulation of hyperpolarization-activated current (I(f)) or L-type Ca(2+) currents (I(Ca,L)), respectively; and 2) direct G(i)-coupled activation of ACh-activated potassium current (I(KACh)). More recent studies, however, have indicated that Ca(2+) cycling by the sarcoplasmic reticulum within SANC (referred to as a Ca(2+) clock) generates rhythmic, spontaneous local Ca(2+) releases (LCR) that are AC-PKA dependent. LCRs activate Na(+)-Ca(2+) exchange (NCX) current, which ignites the surface membrane ion channels to effect an AP. The purpose of the present study was to determine how ChR signaling initiated by a cholinergic agonist, carbachol (CCh), affects AC, cAMP, and PKA or sarcolemmal ion channels and LCRs and how these effects become integrated to generate the net response to a given intensity of ChR stimulation in single, isolated rabbit SANC. The threshold CCh concentration ([CCh]) for BRR was approximately 10 nM, half maximal inhibition (IC(50)) was achieved at 100 nM, and 1,000 nM stopped spontaneous beating. G(i) inhibition by pertussis toxin blocked all CCh effects on BRR. Using specific ion channel blockers, we established that I(f) blockade did not affect BRR at any [CCh] and that I(KACh) activation, evidenced by hyperpolarization, first became apparent at [CCh] > 30 nM. At IC(50), CCh reduced cAMP and reduced PKA-dependent phospholamban (PLB) phosphorylation by approximately 50%. The dose response of BRR to CCh in the presence of I(KACh) blockade by a specific inhibitor, tertiapin Q, mirrored that of CCh to reduced PLB phosphorylation. At IC(50), CCh caused a time-dependent reduction in the number and size of LCRs and a time dependent increase in LCR period that paralleled coincident BRR. The phosphatase inhibitor calyculin A reversed the effect of IC(50) CCh on SANC LCRs and BRR. Numerical model simulations demonstrated that Ca(2+) cycling is integrated into the cholinergic modulation of BRR via LCR-induced activation of NCX current, providing theoretical support for the experimental findings. Thus ChR stimulation-induced BRR is entirely dependent on G(i) activation and the extent of G(i) coupling to Ca(2+) cycling via PKA signaling or to I(KACh): at low [CCh], I(KACh) activation is not evident and BRR is attributable to a suppression of cAMP-mediated, PKA-dependent Ca(2+) signaling; as [CCh] increases beyond 30 nM, a tight coupling between suppression of PKA-dependent Ca(2+) signaling and I(KACh) activation underlies a more pronounced BRR.

Enhanced proliferation of monolayer cultures of embryonic stem (ES) cell-derived cardiomyocytes following acute loss of retinoblastoma.

BACKGROUND: Cardiomyocyte (CM) cell cycle analysis has been impeded because of a reliance on primary neonatal cultures of poorly proliferating cells or chronic transgenic animal models with innate compensatory mechanisms. METHODOLOGY/PRINCIPAL FINDINGS: We describe an in vitro model consisting of monolayer cultures of highly proliferative embryonic stem (ES) cell-derived CM. Following induction with ascorbate and selection with puromycin, early CM cultures are >98% pure, and at least 85% of the cells actively proliferate. During the proliferative stage, cells express high levels of E2F3a, B-Myb and phosphorylated forms of retinoblastoma (Rb), but with continued cultivation, cells stop dividing and mature functionally. This developmental transition is characterized by a switch from slow skeletal to cardiac TnI, an increase in binucleation, cardiac calsequestrin and hypophosphorylated Rb, a decrease in E2F3, B-Myb and atrial natriuretic factor, and the establishment of a more negative resting membrane potential. Although previous publications suggested that Rb was not necessary for cell cycle control in heart, we find following acute knockdown of Rb that this factor actively regulates progression through the G1 checkpoint and that its loss promotes proliferation at the expense of CM maturation. CONCLUSIONS/SIGNIFICANCE: We have established a unique model system for studying cardiac cell cycle progression, and show in contrast to previous reports that Rb actively regulates both cell cycle progression through the G1 checkpoint and maturation of heart cells. We conclude that this in vitro model will facilitate the analysis of cell cycle control mechanisms of CMs.

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 mciroM) 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 Ca(2+) 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 Ca(2+) channel Ca(v)1.2 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.

Signals from embryonic fibroblasts induce adult intestinal epithelial cells to form nestin-positive cells with proliferation and multilineage differentiation capacity in vitro.

The intestinal epithelium has one of the greatest regenerative capacities in the body; however, neither stem nor progenitor cells have been successfully cultivated from the intestine. In this study, we applied an "artificial niche" of mouse embryonic fibroblasts to derive multipotent cells from the intestinal epithelium. Cocultivation of adult mouse and human intestinal epithelium with fibroblast feeder cells led to the generation of a novel type of nestin-positive cells (intestinal epithelium-derived nestin-positive cells [INPs]). Transcriptome analyses demonstrated that mouse embryonic fibroblasts expressed relatively high levels of Wnt/bone morphogenetic protein (BMP) transcripts, and the formation of INPs was specifically associated with an increase in Lef1, Wnt4, Wnt5a, and Wnt/BMP-responsive factors, but a decrease of BMP4 transcript abundance. In vitro, INPs showed a high but finite proliferative capacity and readily differentiated into cells expressing neural, pancreatic, and hepatic transcripts and proteins; however, these derivatives did not show functional properties. In vivo, INPs failed to form chimeras following injection into mouse blastocysts but integrated into hippocampal brain slice cultures in situ. We conclude that the use of embryonic fibroblasts seems to reprogram adult intestinal epithelial cells by modulation of Wnt/BMP signaling to a cell type with a more primitive embryonic-like stage of development that has a high degree of flexibility and plasticity.

Molecular and functional expression of voltage-operated calcium channels during osteogenic differentiation of human mesenchymal stem cells.

UNLABELLED: 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. INTRODUCTION: During osteogenic differentiation, mesenchymal stem cells from human bone marrow (hMSCs) must adopt the calcium handling of terminally differentiated osteoblasts. There is evidence that voltage-operated calcium channels (VOCCs), including L-type calcium channels, are involved in regulation of osteoblast function. We therefore studied whether VOCCs play a critical role during osteogenic differentiation of hMSCs. MATERIALS AND METHODS: Osteogenic differentiation was induced in hMSCs cultured in maintenance medium (MM) by addition of ascorbate, beta-glycerophosphate, and dexamethasone (ODM) and was assessed by measuring alkaline phosphatase activity, expression of osteopontin, osteoprotegerin, RANKL, and mineralization. Expression of Ca2+ channel alpha1 subunits was shown by semiquantitative or single cell RT-PCR. Voltage-activated calcium currents of hMSCs were measured with the whole cell voltage-clamp technique. RESULTS: mRNA for the pore-forming alpha1C and alpha1G subunits of the L-type and T-type Ca2+ channels, respectively, was found in comparable amounts in cells cultured in MM or ODM. The limitation of L-type Ca2+ currents to a subpopulation of hMSCs was confirmed by single cell RT-PCR, where mRNA for the alpha1C subunits was detectable in only 50% of the cells cultured in MM. Dihydropyridine-sensitive L-type Ca2+ currents were found in 13% of cells cultured in MM and in 12% of the cells cultured in ODM. Under MM and ODM culture conditions, the cells positive for L-type Ca2+ currents were significantly larger than cells without Ca2+ currents as deduced from membrane capacitance; thus, current densities were comparable. Addition of the L-type Ca2+ channel blocker nifedipine to the culture media did not influence alkaline phosphatase activity and the extent of mineralization. CONCLUSION: These results suggest that, in the majority of hMSCs, Ca2+ entry through the plasma membrane is mediated by some channels other than VOCCs, and blockade of the L-type Ca2+ channels does not affect early osteogenic differentiation of hMSCs.

Tissue distribution of a human Ca v 1.2 alpha1 subunit splice variant with a 75 bp insertion.

Mesenchymal stem cells from human bone marrow (MSC) express mRNA encoding the L-type Ca2+ channel Ca v 1.2 alpha1 subunit (alpha(1)1.2). We now describe a splice variant including an alternative exon of 75 bp in the region between exons 9 and 10, which we identified in MSC by semi-quantitative RT-PCR. With primers specific for variants including (+9*) or excluding the 75 bp insertion (-9*), we found comparable mRNA expression patterns in MSC and in primary cultures of related connective tissue cells (chondrocytes, osteoblasts and fibroblasts). Since culture conditions might have altered variant expression, we investigated mRNA levels in various native human tissue samples (cartilage, bone, fat, liver, kidney, aorta, bladder, cardiac ventricle and atrium, CNS). We found highest levels of the +9* variant in aorta, containing smooth muscle and connective tissue cells, but the variant was expressed in all tissues. We therefore hypothesized that broad expression of +9* might be linked to the presence of vasculature and/or connective tissue structures, rather than to tissue-specific parenchymal cells (e.g. cardiomyocytes). To test this hypothesis we separated human atrium into a cardiomyocyte-enriched fraction and a cardiomyocyte-depleted fraction. RT-PCR demonstrated significantly larger levels of the +9* variant in the non-cardiomyocyte fraction. The result was even more clear in single cell RT-PCR experiments, where the +9* variant was undetectable in cardiomyocytes but present in non-cardiomyocytes. We conclude that the +9* variant is present in all human tissues investigated so far, and suggest that expression in human atrium is associated with vascular smooth muscle and/or connective tissue cells.

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 alpha1C 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 Ca(2+)-activated K(+) current, conducted by MaxiK channels, due to its high sensitivity to tetraethylammonium (IC(50)= 340 microm) 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 (IC(50)= 0.8 microm). Ba(2+) inward currents, stimulated by 1 microm BayK 8644 were found in a few cells, indicating the expression of functional L-type Ca(2+) 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.