Cav2.3 R-type calcium channels: from its discovery to pathogenic de novo CACNA1E variants: a historical perspective.

So-called pharmacoresistant (R-type) voltage-gated Ca2+ channels are structurally only partially characterized. Most of them are encoded by the CACNA1E gene and are expressed as different Cav2.3 splice variants (variant Cav2.3a to Cav2.3e or f) as the ion conducting subunit. So far, no inherited disease is known for the CACNA1E gene but recently spontaneous mutations leading to early death were identified, which will be brought into focus. In addition, a short historical overview may highlight the development to understand that upregulation during aging, easier activation by spontaneous mutations or lack of bioavailable inorganic cations (Zn2+ and Cu2+) may lead to similar pathologies caused by cellular overexcitation.

Medium-chain fatty acids modulate myocardial function via a cardiac odorant receptor.

Several studies have demonstrated the expression of odorant receptors (OR) in various human tissues and their involvement in different physiological and pathophysiological processes. However, the functional role of ORs in the human heart is still unclear. Here, we firstly report the functional characterization of an OR in the human heart. Initial next-generation sequencing analysis revealed the OR expression pattern in the adult and fetal human heart and identified the fatty acid-sensing OR51E1 as the most highly expressed OR in both cardiac development stages. An extensive characterization of the OR51E1 ligand profile by luciferase reporter gene activation assay identified 2-ethylhexanoic acid as a receptor antagonist and various structurally related fatty acids as novel OR51E1 ligands, some of which were detected at receptor-activating concentrations in plasma and epicardial adipose tissue. Functional investigation of the endogenous receptor was carried out by Ca2+ imaging of human stem cell-derived cardiomyocytes. Application of OR51E1 ligands induced negative chronotropic effects that depended on activation of the OR. OR51E1 activation also provoked a negative inotropic action in cardiac trabeculae and slice preparations of human explanted ventricles. These findings indicate that OR51E1 may play a role as metabolic regulator of cardiac function.

Effective surface-based cryopreservation of human embryonic stem cells by vitrification.

Human embryonic stem cells (hESCs) are candidates for many applications in the areas of regenerative medicine, tissue engineering, basic scientific research as well as pharmacology and toxicology. However, use of hESCs is limited by their sensitivity to freezing and thawing procedures. Hence, this emerging science needs new, reliable preservation methods for the long-term storage of large quantities of functional hESCs remaining pluripotent after post-thawing and culturing. Here, we present a highly efficient, surface based vitrification method for the cryopreservation of large numbers of adherent hESC colonies, using modified cell culture substrates. This technique results in much better post-thaw survival rate compared to cryopreservation in suspension and allows a quick and precise handling and storage of the cells, indicating low differentiation rates.

Disruption of cytoskeletal integrity impairs Gi-mediated signaling due to displacement of Gi proteins.

beta1 integrins play a crucial role as cytoskeletal anchorage proteins. In this study, the coupling of the cytoskeleton and intracellular signaling pathways was investigated in beta1 integrin deficient (-/-) embryonic stem cells. Muscarinic inhibition of the L-type Ca2+ current (ICa) and activation of the acetylcholine-activated K+ current (IK,ACh) was found to be absent in beta1 integrin-/- cardiomyocytes. Conversely, beta adrenoceptor-mediated modulation of ICa was unaffected by the absence of beta1 integrins. This defect in muscarinic signaling was due to defective G protein coupling. This was supported by deconvolution microscopy, which demonstrated that Gi exhibited an atypical subcellular distribution in the beta1 integrin-/- cardiomyocytes. A critical role of the cytoskeleton was further demonstrated using cytochalasin D, which displaced Gi and impaired muscarinic signaling. We conclude that cytoskeletal integrity is required for correct localization and function of Gi-associated signaling microdomains.

Functional characteristics of ES cell-derived cardiac precursor cells identified by tissue-specific expression of the green fluorescent protein.

In contrast to terminally differentiated cardiomyocytes, relatively little is known about the characteristics of mammalian cardiac cells before the initiation of spontaneous contractions (precursor cells). Functional studies on these cells have so far been impossible because murine embryos of the corresponding stage are very small, and cardiac precursor cells cannot be identified because of the lack of cross striation and spontaneous contractions. In the present study, we have used the murine embryonic stem (ES, D3 cell line) cell system for the in vitro differentiation of cardiomyocytes. To identify the cardiac precursor cells, we have generated stably transfected ES cells with a vector containing the gene of the green fluorescent protein (GFP) under control of the cardiac alpha-actin promoter. First, fluorescent areas in ES cell-derived cell aggregates (embryoid bodies [EBs]) were detected 2 d before the initiation of contractions. Since Ca2+ homeostasis plays a key role in cardiac function, we investigated how Ca2+ channels and Ca2+ release sites were built up in these GFP-labeled cardiac precursor cells and early stage cardiomyocytes. Patch clamp and Ca2+ imaging experiments proved the functional expression of the L-type Ca2+ current (ICa) starting from day 7 of EB development. On day 7, using 10 mM Ca2+ as charge carrier, ICa was expressed at very low densities 4 pA/pF. The biophysical and pharmacological properties of ICa proved similar to terminally differentiated cardiomyocytes. In cardiac precursor cells, ICa was found to be already under control of cAMP-dependent phosphorylation since intracellular infusion of the catalytic subunit of protein kinase A resulted in a 1.7-fold stimulation. The adenylyl cyclase activator forskolin was without effect. IP3-sensitive intracellular Ca2+ stores and Ca2+-ATPases are present during all stages of differentiation in both GFP-positive and GFP-negative cells. Functional ryanodine-sensitive Ca2+ stores, detected by caffeine-induced Ca2+ release, appeared in most GFP-positive cells 1-2 d after ICa. Coexpression of both ICa and ryanodine-sensitive Ca2+ stores at day 10 of development coincided with the beginning of spontaneous contractions in most EBs. Thus, the functional expression of voltage-dependent L-type Ca2+ channel (VDCC) is a hallmark of early cardiomyogenesis, whereas IP3 receptors and sarcoplasmic Ca2+-ATPases are expressed before the initiation of cardiomyogenesis. Interestingly, the functional expression of ryanodine receptors/sensitive stores is delayed as compared with VDCC.

Selectivity in signal transduction determined by gamma subunits of heterotrimeric G proteins.

Various heterotrimeric guanine nucleotide-binding proteins have been identified on the basis of the individual subtypes of their alpha subunits. The beta gamma complexes, composed of beta and gamma subunits, remain tightly associated under physiological conditions and have been assumed to constitute a common pool shared among various guanosine triphosphate (GTP)-binding (G) protein heterotrimers. Particular alpha and beta subunit subtypes participate in the signal transduction processes between somatostatin or muscarinic receptors and the voltage-sensitive L-type calcium channel in rat pituitary GH3 cells. Among gamma subunits the gamma 3 subtype was found to be required for coupling of the somatostatin receptor to voltage-sensitive calcium channels, whereas the gamma 4 subtype was found to be required for coupling of the muscarinic receptor to those channels.

Endostatin, the proteolytic fragment of collagen XVIII, induces vasorelaxation.

Collagen XVIII is an important component of the extracellular matrix and is expressed in basement membranes. Its degradation results in the generation of endostatin claimed to possess antiangiogenic activity. To date, only limited knowledge exists with regard to the cellular signaling of this molecule. We show in single-cell measurements using the Ca2+ indicator fura-2 acetoxy methylester (fura-2 AM) and the nitric oxide (NO) indicator 4,5-diaminofluorescein diacetate that application of endostatin (ES) (5 pmol/L, 100 ng/mL) induced Ca2+ spikes and an increase of NO production in human and murine endothelial cells. The NO response was independent of an increase in cytosolic Ca2+ and blocked by the endothelial NO synthase (eNOS) inhibitor NG-nitro-L-arginine methyl ester and by incubation with pertussis toxin known to inhibit G(i/o) proteins. The physiological relevance of this novel signaling pathway of ES was assessed with isometric force measurements in large and small arteries of mouse. Physiological concentrations of ES were found to decrease vascular tone in an endothelium-dependent manner. This occurred via an Arg-Gly-Asp (RGD) peptide-independent pathway through activation of G(i/o) proteins, phosphatidylinositol 3-kinase, Akt, and eNOS. We conclude that the proteolytic matrix fragment ES is a prominent vasorelaxing agent. Because ES is constantly released into the blood, it is a novel regulator of blood pressure and, therefore, represents an interesting pharmacological target.

Characterisation of cholinesterase expression during murine embryonic stem cell differentiation.

It is already established that cholinesterases (ChEs) appear in every embryonic blastema at a very early stage of development, independently from innervation. Embryonic butyrylcholinesterase (BChE) is typically found in cells engaged in proliferation processes, while acetylcholinesterase (AChE) is expressed by cells undergoing morphogenetic processes. In order to better define the regulation of cholinesterases during development, we examined their expressions during in vitro differentiation of two murine embryonic stem cell lines by reverse transcription polymerase chain reaction, histochemistry and enzyme activity measurements. AChE and BChE activity and mRNA were present in the undifferentiated stem cells. To test whether the ChEs expression is regulated during differentiation, we employed the embryoid bodies (EBs) culture method, allowing the cells to differentiate, to then collect them at various stages in culture. Interestingly, phases of differentiation were accompanied by increased AChE transcripts; BChE expression was constant, decreasing at later differentiation stages. Cholinesterase activities showed corresponding patterns, with AChE activity increasing at later stages in culture and BChE slightly decreasing. Histochemistry revealed that AChE and BChE activities were mutually exclusive, being expressed by different cell subpopulations. Thus, we have demonstrated that mouse embryonic stem cells express cholinesterases, the enzymes are functional and their expression is regulated during differentiation. Therefore, it appears that their functions under these conditions are not related to synaptic transmission, but for the developmental processes.

Stem cells in pediatric heart failure.

Pediatric heart failure could be a target for regenerative therapy. Stem cell-based therapy has the potential to provide functional cardiomyocytes. Whereas adult stem cells have shown no or only minimal therapeutic benefit in adults with no evidence of transdifferentiation, embryonic stem cells can differentiate to any cell type, including cardiomyocytes. However, ethical concerns and immunological problems are associated with embryonic stem cells derived from the inner cell mass of blastocysts. Recently, somatic cells could be reprogrammed to a pluripotent state (i.e. induced pluripotent stem cells) with the help of transcription factors. This technique removes ethical and probably also immunological concerns. Nevertheless extensive experimental research will be necessary before cell replacement strategies become clinically applicable. Because the underlying pathophysiology differs significantly with age, caution is warranted extrapolating data obtained in experimental models of cardiac ischemia and clinical studies in adults to the pediatric population. Pediatric heart failure has a good prognosis if causal therapy is possible. However, some forms of congenital heart disease and especially dilated cardiomyopathy still have limited therapeutic options. Almost half of children with symptomatic cardiomyopathy receive a transplant or die within two years. The authors will review the relevant stem cell sources for cell-based treatments. And, given the differences of the underlying diseases between adult and pediatric patients with heart failure, it is contemplated which condition of pediatric patients with heart failure is most likely to benefit and which cell type would be appropriate.

Adult mesenchymal stromal stem cells for therapeutic applications.

Mesenchymal stromal stem cells (MSC) can be found in almost any adult organ. They can be isolated and expanded within several weeks up to hundreds of millions of cells. The cell isolation based on the surface antigen expression may significantly enrich for the desired cell population and reduce the time required for cell expansion. MSC display a unique molecular signature which clearly discriminates them from other stem cell types. MSC can be differentiated into the cells of several lineages. Additionally, the unique biological properties of MSC are mediated by strong immunomodulatory activity and by paracrine mechanisms. Potential therapeutic applications of the cells require clinically compliant protocols for cell isolation and expansion. The therapeutic utility of MSC has been evaluated and found to be useful in several pre-clinical animal models as well as in clinical trials.

A practical guide to the preparation and use of metal ion-buffered systems for physiological research.

Recent recognition that mobile pools of Zn2+ and Cu2+ are involved in the regulation of neuronal, endocrine and other cells has stimulated the development of tools to visualize and quantify the level of free trace metal ions. Most of the methods used to measure or control loosely bound metals require reference media that contain exactly defined free concentrations of the target ions. Despite the central importance of proper metal ion buffering, there is still a lack of international standards and beginners in the field may have difficulties finding a coherent description of how to prepare trace metal ion buffers, especially when experiments are to be performed in multimetal systems. To close this gap, we provide a guide for the design, preparation and use of metal ion-buffered systems that facilitate immediate application under physiologically relevant ionic conditions. Thermodynamic and kinetic concepts of chemical speciation as well as general protocols and specific examples are outlined for the accurate preparation of single- and dual-metal ion buffers. In addition, experiments have been performed with FluoZin-3 to illustrate that metal ion-buffered systems are required for reliable preparation of nanomolar Zn2+ solutions and that dual-metal ion buffers can be used to calibrate suitable fluorescent Zn2+ sensors in the presence of millimolar Ca2+ concentrations. Together, the information provided should sensitize readers to the many potential pitfalls and uncertainties that exist when working with physiologically relevant concentrations of trace metal ions and enable them to formulate their own metal ion buffers for most in vitro applications.

In vitro and in vivo phosphorylation of the Cav2.3 voltage-gated R-type calcium channel.

During the recording of whole cell currents from stably transfected HEK-293 cells, the decline of currents carried by the recombinant human Cav2.3+ß3 channel subunits is related to adenosine triphosphate (ATP) depletion after rupture of the cells. It reduces the number of functional channels and leads to a progressive shift of voltage-dependent gating to more negative potentials (Neumaier F., et al., 2018). Both effects can be counteracted by hydrolysable ATP, whose protective action is almost completely prevented by inhibition of serine/threonine but not tyrosine or lipid kinases. These findings indicate that ATP promotes phosphorylation of either the channel or an associated protein, whereas dephosphorylation during cell dialysis results in run-down. Protein phosphorylation is required for Cav2.3 channel function and could directly influence the normal features of current carried by these channels. Therefore, results from in vitro and in vivo phosphorylation of Cav2.3 are summarized to come closer to a functional analysis of structural variations in Cav2.3 splice variants.

The SRC family kinase LYN redirects B cell receptor signaling in human SLP65-deficient B cell lymphoma cells.

SLP65 represents a critical component in (pre-) B cell receptor signal transduction but is compromised in a subset of pre-B cell-derived acute lymphoblastic leukemia. Based on these findings, we investigated (i.) whether SLP65-deficiency also occurs in mature B cell-derived lymphoma and (ii.) whether SLP65-deficient B cell lymphoma cells use an alternative B cell receptor signaling pathway in the absence of SLP65. Indeed, expression of SLP65 protein was also missing in a fraction of B cell lymphoma cases. While SLP65 is essential for B cell receptor-induced Ca2+ mobilization in normal B cells, B cell receptor engagement in SLP65-deficient as compared to SLP65-reconstituted B cell lymphoma cells resulted in an accelerated yet shortlived Ca2+-signal. B cell receptor engagement of SLP65-deficient lymphoma cells involves SRC kinase activation, which is critical for B cell receptor-dependent Ca2+-mobilisation in the absence but not in the presence of SLP65. As shown by RNA interference, the SRC kinase LYN is required for B cell receptor-induced Ca2+ release in SLP65-deficient B cell lymphoma cells but dispensable after SLP65-reconstitution. B cell receptor engagement in SLP65-deficient B cell lymphoma cells also resulted in tyrosine-phosphorylation of the proliferation- and survival-related MAPK1 and STAT5 molecules, which was sensitive to silencing of the SRC kinase LYN. Inhibition of SRC kinase activity resulted in growth arrest and cell death specifically in SLP65-deficient lymphoma cells. These findings indicate that LYN can short-circuit conventional B cell receptor signaling in SLP65-deficient B cell lymphoma cells and thereby promote activation of survival and proliferation-related molecules.

Loss of annexin A7 leads to alterations in frequency-induced shortening of isolated murine cardiomyocytes.

Annexin A7 has been proposed to function in the fusion of vesicles, acting as a Ca(2+) channel and as Ca(2+)-activated GTPase, thus inducing Ca(2+)/GTP-dependent secretory events. To understand the function of annexin A7, we have performed targeted disruption of the Anxa7 gene in mice. Matings between heterozygous mice produced offspring showing a normal Mendelian pattern of inheritance, indicating that the loss of annexin A7 did not interfere with viability in utero. Mice lacking annexin A7 showed no obvious phenotype and were fertile. To assay for exocytosis, insulin secretion from isolated islets of Langerhans was examined. Ca(2+)-induced and cyclic AMP-mediated potentiation of insulin secretion was unchanged in the absence of annexin A7, suggesting that it is not directly implicated in vesicle fusion. Ca(2+) regulation studied in isolated cardiomyocytes, showed that while cells from early embryos displayed intact Ca(2+) homeostasis and expressed all of the components required for excitation-contraction coupling, cardiomyocytes from adult Anxa7(-/-) mice exhibited an altered cell shortening-frequency relationship when stimulated with high frequencies. This suggests a function for annexin A7 in electromechanical coupling, probably through Ca(2+) homoeostasis.

G-proteins involved in the calcium channel signalling system.

Various mechanisms have been identified by which hormones and neurotransmitters interacting with seven transmembrane alpha-helical spanning segments receptors modulate the activity of ion channels. All of the mechanisms involve heterotrimeric G-proteins; the best documented are hormonal modulations of voltage-dependent Ca2+ channels in cardiac, neuronal and endocrine cells. Recent studies using antisense oligonucleotide probes allow the exact identification of the G-proteins involved in these signal transduction pathways.

Identification and characterization of embryonic stem cell-derived pacemaker and atrial cardiomyocytes.

The aim of this study was to identify and functionally characterize cardiac subtypes during early stages of development. For this purpose, transgenic embryonic stem cells were generated using the alpha-myosin heavy chain promoter driving the expression of the enhanced green fluorescent protein (EGFP). EGFP-positive clusters of cells were first observed as early as 7 days of development, thus, even before the initiation of the contractile activity. Flow cytometry and single-cell fluorescence measurements evidenced large diversities of EGFP intensity. Patch-clamp experiments showed EGFP expression exclusively in pacemaker and atrial but not ventricular cells. The highest fluorescence intensities were detected in pacemaker-like cardiomyocytes. In accordance, multielectrode-array recordings of whole embryoid bodies confirmed that the pacemaker center coincided with strongly EGFP-positive areas. The cardiac subtypes displayed already at this early stage differential characteristics of electrical activity and ion channel expression. Thus, quantitation of the alpha-myosin heavy chain driven reporter gene expression allows identification and functional characterization of early cardiac subtypes.

Embryonic stem cells differentiate in vitro into cardiomyocytes representing sinusnodal, atrial and ventricular cell types.

Pluripotent embryonic stem cells (ESC, ES cells) of line D3 were differentiated in vitro and via embryo-like aggregates (embryoid bodies) of defined cell number into spontaneously beating cardiomyocytes. By using RT-PCR technique, alpha- and beta-cardiac myosin heavy chain (MHC) genes were found to be expressed in embryoid bodies of early to terminal differentiation stages. The exclusive expression of the beta-cardiac MHC gene detected in very early differentiated embryoid bodies proved to be dependent on the number of ES cells developing in the embryoid body. Cardiomyocytes enzymatically isolated from embryoid body outgrowths at different stages of development were further characterized by immunocytological and electrophysiological techniques. All cardiomyocytes appeared to be positive in immunofluorescence assays with monoclonal antibodies against cardiac-specific alpha-cardiac MHC, as well as muscle-specific sarcomeric myosin heavy chain and desmin. The patch-clamp technique allowed a more detailed characterization of the in vitro differentiated cardiomyocytes which were found to represent phenotypes corresponding to sinusnode, atrium or ventricle of the heart. The cardiac cells of early differentiated stage expressed pacemaker-like action potentials similar to those described for embryonic cardiomyocytes. The action potentials of terminally differentiated cells revealed shapes, pharmacological characteristics and hormonal regulation inherent to adult sinusnodal, atrial or ventricular cells. In cardiomyocytes of intermediate differentiation state, action potentials of very long duration (0.3-1 s) were found, which may represent developmentally controlled transitions between different types of action potentials. Therefore, the presented ES cell differentiation system permits the investigation of commitment and differentiation of embryonic cells into the cardiomyogenic lineage in vitro.

Differentiation of pluripotent embryonic stem cells into the neuronal lineage in vitro gives rise to mature inhibitory and excitatory neurons.

Embryonic stem (ES) cells represent a suitable model to analyze cell differentiation processes in vitro. Here, we report that pluripotent ES cells of the line BLC 6 differentiate in vitro into neuronal cells possessing the complex electrophysiological and immunocytochemical properties of postmitotic nerve cells. In the course of differentiation BLC 6-derived neurons differentially express voltage-dependent (K+, Na+, Ca2+) and receptor-operated (GABAA, glycine, AMPA, NMDA receptors) ionic channels. They generate fast Na(+)-driven action potentials and are functionally coupled by inhibitory (GABAergic) and excitatory (glutamatergic) synapses as revealed by measurements of postsynaptic currents. Moreover, BLC 6-derived neurons express neuron-specific cytoskeletal, cell adhesion and synaptic vesicle proteins and exhibit a Ca(2+)-dependent GABA secretion. Thus, the ES cell model enables the investigation of cell lineage determination and signaling mechanisms in the developing nervous system from a pluripotential stem cell to a differentiated postmitotic neuron. The in vitro differentiation of neurons from ES cells may be an excellent approach to study by targeted gene disruption a variety of neuronal functions.

Activation of p90RSK and growth stimulation of multicellular tumor spheroids are dependent on reactive oxygen species generated after purinergic receptor stimulation by ATP.

Mitogenic stimulation by growth factors may be mediated through intracellular reactive oxygen species (ROS) acting as signaling molecules. Incubation of multicellular prostate tumor spheroids with adenosine 5' triphosphate (ATP) dose-dependently stimulated tumor growth. ATP, uridine 5'-triphosphate (UTP), adenosine 5'-diphosphate (ADP), and 2-methylthio-ATP (2-MeS-ATP) increased intracellular ROS levels significantly. ROS generation by ATP was inhibited by the P2 receptor antagonist suramin, by the reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitors diphenylene iodonium chloride (DPI) and 4-(2-aminoethyl) benzenesulfonylfluoride (AEBSF), as well as by the Ca2+-dependent phospholipase A2 (PLA2) inhibitors indomethacin and methyl arachidonyl fluorophosphonate (MAFP). The generation of ROS was dependent on the intracellular Ca2+ response evoked by ATP. Exogenous ATP activated the extracellular signal-regulated kinase 1/2 (ERK1/2) mitogen-activated protein kinase (MAPK) pathway, which was blunted by the MAPK/ERK kinase 1/2 (MEK1/2) antagonist PD98059. The radical scavengers vitamin E, dimethyl thiourea (DMTU), and N-acetyl cysteine (NAC) failed to inhibit ERK1/2 activation but abolished p90 ribosomal S6 kinase (p90RSK) activation downstream of ERK1/2, as well as the growth stimulation of tumor spheroids. Our data indicate that p90RSK downstream of ERK1/2 is the molecular target for ROS generated through stimulation of purinergic receptors by ATP.

Ras proteins activate calcium channels in neuronal cells.

Ras (p21) proteins are involved in the control of cell growth and differentiation, but the mechanism by which they exert these effects is not yet known. Here we present evidence that c-Ha-ras (p21(Gly-12)) and its oncogenic mutant T24-ras (p21(Val-12)) selectively induce omega-conotoxin and dihydropyridine-sensitive Ca2+ currents within a few hours after introduction into the cytoplasm of neuroblastoma x glioma hybrid cells. Whereas control cells exhibited a mean Ca2+ current of 250 pA, it amounted to 730 pA in cells pretreated with ras protein. In cells loaded with p21(Gly-12), the effect occurred after 2 hours and was terminated after 8 hours. In contrast, introduction of p21(Val-12) resulted in a prolonged delay (6 hours) of the effect which lasted for more than 24 hours. When ras proteins were preactivated with the non-hydrolysable GTP analog GppNHp, the time courses of both p21(Gly-12) and p21(Val-12) effects were fast and sustained, suggesting that in intact cells (i) the GDP/GTP exchange is faster for p21(Gly-12) compared to p21(Val-12) and (ii) inactivation of p21(Gly-12) is mediated by GAP-induced GTPase activity. T-type Ca2+ currents and K+ currents were unaffected by ras proteins.