Ca Signaling in Cardiovascular Fibroblasts.

Fibrogenesis is a physiological process required for wound healing and tissue repair. It is induced by activation of quiescent fibroblasts, which first proliferate and then change their phenotype into migratory, contractile myofibroblasts. Myofibroblasts secrete extracellular matrix proteins, such as collagen, to form a scar.

Receptor-specific regulation of atrial GIRK channel activity by different Ca-dependent PKC isoforms.

G Protein-activated K channels (GIRK) channels are inhibited by depletion of PtdIns(4,5)P(PIP), and/or channel phosphorylation by proteinkinase C (PKC). By using FRET-based biosensors, expressed in HEK293 cells or in atrial myocytes, we quantified receptor-specific G-coupled receptor (GPCR) signalling on the level of phospholipase C (PLC) activation by monitoring PIP-depletion and diacylglycerol (DAG) formation. Simultaneous voltage-clamp experiments on GIRK channel activity were performed as a functional readout for G-coupled α- and ET-receptor-induced signalling.

Biased signaling of Ca-sensing receptors in cardiac myocytes regulates GIRK channel activity.

Ca-sensing receptors (CaSRs) belong to the class C of G protein-coupled receptors and are activated by extracellular Ca. CaSRs display biased G protein signaling by coupling to different classes of heterotrimeric G proteins depending on agonist and cell type. In this study we used fluorescent biosensors to directly analyze G protein coupling to CaSRs and downstream signaling in living cells.

The allosteric site regulates the voltage sensitivity of muscarinic receptors.

Muscarinic receptors (M-Rs) for acetylcholine (ACh) belong to the class A of G protein-coupled receptors. M-Rs are activated by orthosteric agonists that bind to a specific site buried in the M-R transmembrane helix bundle. In the active conformation, receptor function can be modulated either by allosteric modulators, which bind to the extracellular receptor surface or by the membrane potential via an unknown mechanism.

Receptor Species-dependent Desensitization Controls KCNQ1/KCNE1 K+ Channels as Downstream Effectors of Gq Protein-coupled Receptors.

Activation of G protein-coupled receptors (GPCRs) might induce divergent cellular responses, related to receptor-specific activation of different branches of the G signaling pathway. Receptor-specific desensitization provides a mechanism of effector modulation by restricting the spatiotemporal activation of signaling components downstream of G We quantified signaling events downstream of GPCR activation with FRET-based biosensors in CHO and HEK 293 cells. KCNQ1/KCNE1 channels (I) were measured as a functional readout of receptor-specific activation.

The mode of agonist binding to a G protein-coupled receptor switches the effect that voltage changes have on signaling.

Signaling by many heterotrimeric guanine nucleotide-binding protein (G protein)-coupled receptors (GPCRs) is either enhanced or attenuated by changes in plasma membrane potential. To identify structural correlates of the voltage sensitivity of GPCR signaling, we chose muscarinic acetylcholine receptors (the M1, M3, and M5 isoforms) as a model system. We combined molecular docking analysis with Förster resonance energy transfer (FRET)-based assays that monitored receptor activity under voltage clamp conditions.

Membrane Potential Controls the Efficacy of Catecholamine-induced β1-Adrenoceptor Activity.

G protein-coupled receptors (GPCRs) are membrane-located proteins and, therefore, are exposed to changes in membrane potential (V(M)) in excitable tissues. These changes have been shown to alter receptor activation of certain Gi-and Gq-coupled GPCRs. By means of a combination of whole-cell patch-clamp and Förster resonance energy transfer (FRET) in single cells, we demonstrate that the activation of the Gs-coupled β1-adrenoreceptor (β1-AR) by the catecholamines isoprenaline (Iso) and adrenaline (Adr) is regulated by V(M).

Early subcellular Ca2+ remodelling and increased propensity for Ca2+ alternans in left atrial myocytes from hypertensive rats.

Aims: Hypertension is a major risk factor for atrial fibrillation. We hypothesized that arterial hypertension would alter atrial myocyte calcium (Ca2+) handling and that these alterations would serve to trigger atrial tachyarrhythmias.

Methods And Results: Left atria or left atrial (LA) myocytes were isolated from spontaneously hypertensive rats (SHR) or normotensive Wistar-Kyoto (WKY) controls.

Dynamics of Gαq-protein-p63RhoGEF interaction and its regulation by RGS2.

Some G-protein-coupled receptors regulate biological processes via Gα12/13- or Gαq/11-mediated stimulation of RhoGEFs (guanine-nucleotide-exchange factors). p63RhoGEF is known to be specifically activated by Gαq/11 and mediates a major part of the acute response of vascular smooth muscle cells to angiotensin II treatment. In order to gain information about the dynamics of receptor-mediated activation of p63RhoGEF, we developed a FRET-based assay to study interactions between Gαq-CFP and Venus-p63RhoGEF in single living cells.

Dynamics of Gαi1 interaction with type 5 adenylate cyclase reveal the molecular basis for high sensitivity of Gi-mediated inhibition of cAMP production.

Many physiological and pathophysiological processes are regulated by cAMP. Different therapies directly or indirectly influence the cellular concentration of this second messenger. A wide variety of receptors either activates or inhibits adenylate cyclases in order to induce proper physiological responses.

Voltage regulates adrenergic receptor function.

The present study demonstrates that agonist-mediated activation of α2A adrenergic receptors (α(2A)AR) is voltage-dependent. By resolving the kinetics of conformational changes of α(2A)AR at defined membrane potentials, we show that negative membrane potentials in the physiological range promote agonist-mediated activation of α(2A)AR. We discovered that the conformational change of α(2A)AR by voltage is independent from receptor-G protein docking and regulates receptor signaling, including β-arrestin binding, activation of G proteins, and G protein-activated inwardly rectifying K(+) currents.

Activation of NFATc1 is directly mediated by IP3 in adult cardiac myocytes.

The Ca(2+)-sensitive nuclear factor of activated T cell (NFAT) transcription factors are implicated in cardiac development and cellular remodeling associated with cardiac disease. In adult myocytes it is not resolved what specific Ca(2+) signals control the activity of different NFAT isoforms in an environment that undergoes large changes of intracellular Ca(2+) concentration with every heart beat. Cardiac myocytes possess the complete inositol 1,4,5-trisphosphate (IP(3))/Ca(2+)-signaling cassette; however, its physiological and pathological significance has been a matter of ongoing debate.

A fluorescence-based assay to monitor transcriptional activity of NFAT in living cells.

Ca(2+)-sensitive NFAT (nuclear factor of activated T-cells) transcription factors are implicated in many pathophysiological processes in different cell types. The precise control of activation varies with NFAT isoform and cell type. Here we present feasibility of an in vivo assay (NFAT-RFP) that reports transcriptional activity of NFAT via expression of red fluorescent protein (RFP) in individual cells.

Isoform- and tissue-specific regulation of the Ca(2+)-sensitive transcription factor NFAT in cardiac myocytes and heart failure.

Nuclear factors of activated T cells (NFATs) are Ca(2+)-sensitive transcription factors that have been implicated in hypertrophy, heart failure (HF), and arrhythmias. Cytosolic NFAT is activated by dephosphorylation by the Ca(2+)-sensitive phosphatase calcineurin, resulting in translocation to the nucleus, which is opposed by kinase activity, rephosphorylation, and nuclear export. Four different NFAT isoforms are expressed in the heart.

Regulation of nuclear factor of activated T cells (NFAT) in vascular endothelial cells.

Proteins of the NFAT family (nuclear factor of activated T cells) are Ca(2+)-sensitive transcription factors, which are involved in hypertrophic cardiovascular remodeling. Activation and nuclear translocation is mediated by dephosphorylation by the Ca(2+)-sensitive phosphatase calcineurin (CaN). We identified Ca(2+) signals that induced nuclear translocation of NFAT in cultured calf pulmonary artery endothelial (CPAE) cells using confocal fluorescence microscopy to measure simultaneously [Ca(2+)](i) and subcellular localization of NFAT-GFP (isoforms NFATc1 and NFATc3).

Adenovirus-mediated delivery of short hairpin RNA (shRNA) mediates efficient gene silencing in terminally differentiated cardiac myocytes.

RNA interference (RNAi) represents the most frequently utilized technique to analyze proteins by loss of function assays. Protein synthesis is impaired by sequence-specific degradation of mRNA, which is triggered by short (19-28 nt) silencing RNAs (siRNA). Efficient gene silencing using RNAi has been demonstrated in numerous cell lines and primary cultured cells.

G protein-activated (GIRK) current in rat ventricular myocytes is masked by constitutive inward rectifier current (I(K1)).

Inwardly-rectifying K+ channel subunits are not homogenously expressed in different cardiac tissues. In ventricular myocytes (VM) the background current-voltage relation is dominated by I(K1), carried by channels composed of Kir2.x subunits, which is less important in atrial myocytes (AM).

Generation of a constitutive Na+-dependent inward-rectifier current in rat adult atrial myocytes by overexpression of Kir3.4.

Apart from gating by interaction with betagamma subunits from heterotrimeric G proteins upon stimulation of appropriate receptors, Kir.3 channels have been shown to be gated by intracellular Na+. However, no information is available on how Na+-dependent gating affects endogenous Kir3.

Gene silencing in adult rat cardiac myocytes in vitro by adenovirus-mediated RNA interference.

RNA interference (RNAi) by short double stranded RNA (siRNA) represents an efficient and frequently used tool for gene silencing to study gene function. Whereas efficient ablation of genes has been demonstrated in neonatal cardiac myocytes, thus far information on successful application of this technique in adult cardiac myocytes (ACM), a standard experimental model in cardiac physiology and pathophysiology, is sparse. Here we demonstrate efficient ablation of a transgene encoding for enhanced green fluorescent protein (EGFP) and a cell specific endogenous gene encoding for an inward-rectifier channel subunit (Kir2.

Voltage dependence of ATP-dependent K+ current in rat cardiac myocytes is affected by IK1 and IK(ACh).

In this study we have investigated the voltage dependence of ATP-dependent K+ current (I(K(ATP))) in atrial and ventricular myocytes from hearts of adult rats and in CHO cells expressing Kir6.2 and SUR2A. The current-voltage relation of 2,4-dinitrophenole (DNP) -induced I(K(ATP)) in atrial myocytes and expressed current in CHO cells was linear in a voltage range between 0 and -100 mV.

Acute desensitization of GIRK current in rat atrial myocytes is related to K+ current flow.

We have investigated the acute desensitization of acetylcholine-activated GIRK current (I(K(ACh))) in cultured adult rat atrial myocytes. Acute desensitization of I(K(ACh)) is observed as a partial relaxation of current with a half-time of < 5 s when muscarinic M2 receptors are stimulated by a high concentration (> 2 micromol l(-1)) of ACh. Under this condition experimental manoeuvres that cause a decrease in the amplitude of I(K(ACh)), such as partial block of M2 receptors by atropine, intracellular loading with GDP-beta-S, or exposure to Ba2+, caused a reduction in desensitization.