Angiotensin receptors and α1B-adrenergic receptors regulate native IK(ACh) and phosphorylation-deficient GIRK4 (S418A) channels through different PKC isoforms.

Signaling of G protein-activated inwardly rectifying K+ (GIRK) channels is an important mechanism of the parasympathetic regulation of the heart rate and cardiac excitability. GIRK channels are inhibited during stimulation of Gq-coupled receptors (GqPCRs) by depletion of phosphatidyl-4,5-bisphosphate (PIP2) and/or channel phosphorylation by protein kinase C (PKC). The GqPCR-dependent modulation of GIRK currents in terms of specific PKC isoform activation was analyzed in voltage-clamp experiments in rat atrial myocytes and in CHO or HEK 293 cells. By using specific PKC inhibitors, we identified the receptor-activated PKC isoforms that contribute to phenylephrine- and angiotensin-induced GIRK channel inhibition. We demonstrate that the cPKC isoform PKCα significantly contributes to GIRK inhibition during stimulation of wildtype α1B-adrenergic receptors (α1B-ARs). Deletion of the α1B-AR serine residues S396 and S400 results in a preferential regulation of GIRK activity by PKCß. As a novel finding, we report that the AT1-receptor-induced GIRK inhibition depends on the activation of the nPKC isoform PKCε whereas PKCα and PKCß do not mainly participate in the angiotensin-mediated GIRK reduction. Expression of the dominant negative (DN) PKCε prolonged the onset of GIRK inhibition and significantly reduced AT1-R desensitization, indicating that PKCε regulates both GIRK channel activity and the strength of the receptor signal via a negative feedback mechanism. The serine residue S418 represents an important phosphorylation site for PKCε in the GIRK4 subunit. To analyze the functional impact of this PKC phosphorylation site for receptor-specific GIRK channel modulation, we monitored the activity of a phosphorylation-deficient (GIRK4 (S418A)) GIRK4 channel mutant during stimulation of α1B-ARs or AT1-receptors. Mutation of S418 did not impede α1B-AR-mediated GIRK inhibition, suggesting that S418 within the GIRK4 subunit is not subject to PKCα-induced phosphorylation. Furthermore, activation of angiotensin receptors induced pronounced GIRK4 (S418A) channel inhibition, excluding that this phosphorylation site contributes to the AT1-R-induced GIRK reduction. Instead, phosphorylation of S418 has a facilitative effect on GIRK activity that was abolished in the GIRK4 (S418A) mutant. To summarize, the present study shows that the receptor-dependent regulation of atrial GIRK channels is attributed to the GqPCR-specific activation of different PKC isoforms. Receptor-specific activated PKC isoforms target distinct phosphorylation sites within the GIRK4 subunit, resulting in differential regulation of GIRK channel activity with either facilitative or inhibitory effects on GIRK currents.

Human skeletal muscle organoids model fetal myogenesis and sustain uncommitted PAX7 myogenic progenitors.

In vitro culture systems that structurally model human myogenesis and promote PAX7+ myogenic progenitor maturation have not been established. Here we report that human skeletal muscle organoids can be differentiated from induced pluripotent stem cell lines to contain paraxial mesoderm and neuromesodermal progenitors and develop into organized structures reassembling neural plate border and dermomyotome. Culture conditions instigate neural lineage arrest and promote fetal hypaxial myogenesis toward limb axial anatomical identity, with generation of sustainable uncommitted PAX7 myogenic progenitors and fibroadipogenic (PDGFRa+) progenitor populations equivalent to those from the second trimester of human gestation. Single-cell comparison to human fetal and adult myogenic progenitor /satellite cells reveals distinct molecular signatures for non-dividing myogenic progenitors in activated (CD44High/CD98+/MYOD1+) and dormant (PAX7High/FBN1High/SPRY1High) states. Our approach provides a robust 3D in vitro developmental system for investigating muscle tissue morphogenesis and homeostasis.

Humans contains around 650 skeletal muscles which allow the body to move around and maintain its posture. Skeletal muscles are made up of individual cells that bundle together into highly organized structures. If this group of muscles fail to develop correctly in the embryo and/or fetus, this can lead to muscular disorders that can make it painful and difficult to move. One way to better understand how skeletal muscles are formed, and how this process can go wrong, is to grow them in the laboratory. This can be achieved using induced pluripotent stem cells (iPSCs), human adult cells that have been 'reprogrammed' to behave like cells in the embryo that can develop in to almost any cell in the body. The iPSCs can then be converted into specific cell types in the laboratory, including the cells that make up skeletal muscle. Here, Mavrommatis et al. created a protocol for developing iPSCs into three-dimensional organoids which resemble how cells of the skeletal muscle look and arrange themselves in the fetus. To form the skeletal muscle organoid, Mavrommatis et al. treated iPSCs that were growing in a three-dimensional environment with various factors that are found early on in development. This caused the iPSCs to organize themselves in to embryonic and fetal structures that will eventually give rise to the parts of the body that contain skeletal muscle, such as the limbs. Within the organoid were cells that produced Pax7, a protein commonly found in myogenic progenitors that specifically mature into skeletal muscle cells in the fetus. Pax 7 is also present in 'satellite cells' that help to regrow damaged skeletal muscle in adults. Indeed, Mavrommatis et al. found that the myogenic progenitors produced by the organoid were able to regenerate muscle when transplanted in to adult mice. These findings suggest that this organoid protocol can generate cells that will give rise to skeletal muscle. In the future, these lab-grown progenitors could potentially be created from cells isolated from patients and used to repair muscle injuries. The organoid model could also provide new insights in to how skeletal muscles develop in the fetus, and how genetic mutations linked with muscular disorders disrupt this process.

CRISPR/Cas9 Genome Editing in LGMD2A/R1 Patient-Derived Induced Pluripotent Stem and Skeletal Muscle Progenitor Cells.

Large numbers of Calpain 3 (CAPN3) mutations cause recessive forms of limb-girdle muscular dystrophy (LGMD2A/LGMDR1) with selective atrophy of the proximal limb muscles. We have generated induced pluripotent stem cells (iPSC) from a patient with two mutations in exon 3 and exon 4 at the calpain 3 locus (W130C, 550delA). Two different strategies to rescue these mutations are devised: (i) on the level of LGMD2A-iPSC, we combined CRISPR/Cas9 genome targeting with a FACS and Tet transactivator-based biallelic selection strategy, which resulted in a new functional chimeric exon 3-4 without the two CAPN3 mutations. (ii) On the level of LGMD2A-iPSC-derived CD82+/Pax7+ myogenic progenitor cells, we demonstrate CRISPR/Cas9 mediated rescue of the highly prevalent exon 4 CAPN3 mutation. The first strategy specifically provides isogenic LGMD2A corrected iPSC for disease modelling, and the second strategy can be further elaborated for potential translational approaches.

PKC-isoform specific regulation of receptor desensitization and KCNQ1/KCNE1 K+ channel activity by mutant α1B-adrenergic receptors.

Activation of a specific protein kinase C (PKC) isoform during stimulation of Gq protein-coupled receptors (GqPCRs) is determined by homologous receptor desensitization that controls the spatiotemporal formation of downstream Gq signalling molecules. Furthermore, GqPCR-activated PKC isoforms specifically regulate receptor activity via a negative feedback mechanism. In the present study, we investigated the contribution of several phosphorylation sites in the α1B-adrenergic receptor (α1B-AR) for PKC and G protein coupled receptor kinase 2 (GRK2) to homologous receptor desensitization and effector modulation. We analyzed signalling events downstream to human wildtype α1B-ARs and α1B-ARs lacking PKC or GRK2 phosphorylation sites (Δ391-401, α1B-ΔPKC-AR and Δ402-520, α1B-ΔGRK-AR) by means of FRET-based biosensors in HEK293 that served as online-assays of receptor activity. K+ currents through KCNQ1/KCNE1 channels (IKs), which are regulated by both phosphatidylinositol 4,5-bisphosphate (PIP2)-depletion and/or phosphorylation by PKC, were measured as a functional readout of wildtype and mutant α1B-AR receptor activity. As a novel finding, we provide evidence that deletion of PKC and GRK2 phosphorylation sites in α1B-ARs abrogates the contribution of PKCα to homologous receptor desensitization. Instead, the time course of mutant receptor activity was specifically modulated by PKCß. Mutant α1B-ARs displayed pronounced homologous receptor desensitization that was abolished by PKCß-specific pharmacological inhibitors. IKs modulation during stimulation of wildtype and mutant α1B-ARs displayed transient inhibition and current facilitation after agonist withdrawal with reduced capability of mutant α1B-ARs to induce IKs inhibition. Pharmacological inhibition of the PKCß isoform did not augment IKs reduction by mutant α1B-ARs, but shifted IKs modulation towards current facilitation. Coexpression of an inactive (dominant-negative) PKCδ isoform (DN-PKCδ) abolished IKs facilitation in α1B-ΔGRK-AR-expressing cells, but not in α1B-ΔPKC-AR-expressing cells. The data indicate that the differential modulation of IKs activity by α1B-ΔGRK- and α1B-ΔPKC-receptors is attributed to the activation of entirely distinct novel PKC isoforms. To summarize, specific phosphorylation sites within the wildtype and mutant α1B-adrenergic receptors are targeted by different PKC isoforms, resulting in differential regulation of receptor desensitization and effector function.

Receptor-specific regulation of atrial GIRK channel activity by different Ca2+-dependent PKC isoforms.

G Protein-activated K+ channels (GIRK) channels are inhibited by depletion of PtdIns(4,5)P2(PIP2), 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 Gq-coupled receptor (GqPCR) signalling on the level of phospholipase C (PLC) activation by monitoring PIP2-depletion and diacylglycerol (DAG) formation. Simultaneous voltage-clamp experiments on GIRK channel activity were performed as a functional readout for Gq-coupled α1B- and ET-receptor-induced signalling. GqPCR-induced fast inhibition of GIRK channel activity is mediated by depletion of PIP2, whereas phosphorylation of GIRK channels results in delayed, but effective GIRK current inhibition. We demonstrate a receptor-induced inhibitory component on GIRK activity that is independent of PIP2-depletion, but attributed to the activation of Ca2+-dependent PKC isoforms. As a novel finding, we demonstrate receptor-dependent differences in GIRK inhibition according to receptor-specific activation of the Ca2+-dependent PKC isoforms PKCα and PKCß. Pharmacological inhibition of PKCα, but not of PKCß, abolishes GIRK inhibition induced by stimulation of α1B-receptors. In contrast, ET-R-induced reduction of GIRK activity is sensitive to pharmacological block of PKCß, but not of PKCα. Coexpression of α1B-receptors (or ETB-R) and PKCα (or PKCß) in HEK 293 cells increased homologous receptor desensitization as indicated by a rapid decline of the CKAR FRET signal monitoring receptor activity. These data suggest that receptor-species dependent differences in PKC isoform activation regulate both GIRK channel activity and the strength of the receptor signal via a negative feedback mechanism.

Biased signaling of Ca2+-sensing receptors in cardiac myocytes regulates GIRK channel activity.

Ca2+-sensing receptors (CaSRs) belong to the class C of G protein-coupled receptors and are activated by extracellular Ca2+. 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. In HEK 293 cells, CaSRs displayed biased signaling: elevation of extracellular Ca2+ or application of the alternative agonist spermine caused activation of Gi- and Gq-proteins. Adult cardiac myocytes express endogenous CaSRs, which have been implicated in regulating Ca2+ signaling and contractility. Biased signaling of CaSRs has not been investigated in these cells. To evaluate efficiencies of Gi- and Gq-signaling via CaSRs in rat atrial myocytes, we measured G protein-activated K+ (GIRK) channels. Activation of GIRK requires binding of Gßγ subunits released from Gi proteins, whereas Gq-signaling results in inhibition of GIRK channel activity. Stimulation of CaSRs by Ca2+ or spermine failed to directly activate Gi and GIRK channels. When GIRK channels were pre-activated via endogenous M2 receptors, stimulation of CaSRs caused pronounced inhibition of GIRK currents. This effect was specific to CaSR activation: GIRK current inhibition was sensitive to NPS-2143, a negative allosteric modulator of CaSRs, and abrogated by FR900359, a direct inhibitor of Gq. GIRK current inhibition was also sensitive to the PKC inhibitor chelerythrine, suggesting that following activation of CaSR and Gq, GIRK currents are modulated by PKC phosphorylation. We conclude from this data that cardiac CaSRs do not activate Gi and affect GIRK currents preferentially via the Gq/PKC pathway.

Familial Sinus Node Disease Caused by a Gain of GIRK (G-Protein Activated Inwardly Rectifying K+ Channel) Channel Function.

BACKGROUND: Inherited forms of sinus node dysfunction (SND) clinically include bradycardia, sinus arrest, and chronotropic incompetence and may serve as disease models to understand sinus node physiology and impulse generation. Recently, a gain-of-function mutation in the G-protein gene GNB2 led to enhanced activation of the GIRK (G-protein activated inwardly rectifying K+ channel). Thus, human cardiac GIRK channels are important for heart rate regulation and subsequently, genes encoding their subunits Kir3.1 and Kir3.4 ( KCNJ3 and KCNJ5) are potential candidates for inherited SND in human. METHODS: We performed a combined approach of targeted sequencing of KCNJ3 and KCNJ5 in 52 patients with idiopathic SND and subsequent whole exome sequencing of additional family members in a genetically affected patient. A putative novel disease-associated gene variant was functionally analyzed by voltage-clamp experiments using various heterologous cell expression systems (Xenopus oocytes, CHO cells, and rat atrial cardiomyocytes). RESULTS: In a 3-generation family with SND we identified a novel variant in KCNJ5 which leads to an amino acid substitution (p.Trp101Cys) in the first transmembrane domain of the Kir3.4 subunit of the cardiac GIRK channel. The identified variant cosegregated with the disease in the family and was absent in the Exome Variant Server and Exome Aggregation Consortium databases. Expression of mutant Kir3.4 (±native Kir3.1) in different heterologous cell expression systems resulted in increased GIRK currents ( IK,ACh) and a reduced inward rectification which was not compensated by intracellular spermidine. Moreover, in silico modeling of heterotetrameric mutant GIRK channels indicates a structurally altered binding site for spermine. CONCLUSIONS: For the first time, an inherited gain-of-function mutation in the human GIRK3.4 causes familial human SND. The increased activity of GIRK channels is likely to lead to a sustained hyperpolarization of pacemaker cells and thereby reduces heart rate. Modulation of human GIRK channels may pave a way for further treatment of cardiac pacemaking.

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

Activation of Gq protein-coupled receptors (GqPCRs) might induce divergent cellular responses, related to receptor-specific activation of different branches of the Gq signaling pathway. Receptor-specific desensitization provides a mechanism of effector modulation by restricting the spatiotemporal activation of signaling components downstream of Gq We quantified signaling events downstream of GqPCR activation with FRET-based biosensors in CHO and HEK 293 cells. KCNQ1/KCNE1 channels (IKs) were measured as a functional readout of receptor-specific activation. Activation of muscarinic M1 receptors (M1-Rs) caused robust and reversible inhibition of IKs. In contrast, activation of α1B-adrenergic receptors (α1B-ARs) induced transient inhibition of IKs, which turned into delayed facilitation after agonist withdrawal. As a novel finding, we demonstrate that GqPCR-specific kinetics of IKs modulation are determined by receptor-specific desensitization, evident at the level of Gαq activation, phosphatidylinositol 4,5-bisphosphate (PIP2) depletion, and diacylglycerol production. Sustained IKs inhibition during M1-R stimulation is attributed to robust membrane PIP2 depletion, whereas the rapid desensitization of α1B-AR delimits PIP2 reduction and augments current activation by protein kinase C (PKC). Overexpression of Ca2+-independent PKCδ did not affect the time course of α1B-AR-induced diacylglycerol formation, excluding a contribution of PKCδ to α1B-AR desensitization. Pharmacological inhibition of Ca2+-dependent PKC isoforms abolished fast α1B receptor desensitization and augmented IKs reduction, but did not affect IKs facilitation. These data indicate a contribution of Ca2+-dependent PKCs to α1B-AR desensitization, whereas IKs facilitation is induced by Ca2+-independent PKC isoforms. In contrast, neither inhibition of Ca2+-dependent/Ca2+-independent isoforms nor overexpression of PKCδ induced M1 receptor desensitization, excluding a contribution of PKC to M1-R-induced IKs modulation.

NCI-H295R cell line as in vitro model of hyperaldosteronism lacks functional KCNJ5 (GIRK4; Kir3.4) channels.

As a major cause of aldosterone producing adenomas, numerous gain-of-function mutations in the KCNJ5 gene (encoding the K(+) channel subunit GIRK4) have been identified. The human adrenocortical carcinoma cell line NCI-H295R is the most frequently used cellular model for in vitro studies related to regulation of aldosterone-synthesis. Because of the undefined role of KCNJ5 (GIRK4) in regulating synthesis of aldosterone, we aimed at identifying basal and G protein-activated GIRK4 currents in this paradigmatic cell line. The GIRK-specific blocker Tertiapin-Q did not affect basal current. Neither loading of the cells with GTP-γ-S via the patch-clamp pipette nor agonist stimulation of an infected A1-adenosine receptor resulted in activation of GIRK current. In cells co-infected with KCNJ5, robust activation of basal and adenosine-activated inward-rectifying current was observed. Although GIRK4 protein can be detected in Western blots of H295R homogenates, we suggest that GIRK4 in aldosterone-producing cells does not form functional G(ßγ)-activated channels.

Synergistic modulation of KCNQ1/KCNE1 K(+) channels (IKs) by phosphatidylinositol 4,5-bisphosphate (PIP2) and [ATP]i.

Cardiac KCNQ1/KCNE1 channels (IKs) are dependent on the concentration of membrane phosphatidylinositol-4,5-bisphosphate (PIP2) and on cytosolic ATP by two distinct mechanisms. In this study we measured IKs and FRET between PH-PLCδ-based fluorescent PIP2 sensors in a stable KCNQ1/KCNE1 CHO cell line. Effects of activating either a muscarinic M3 receptor or the switchable phosphatase Ci-VSP on IKs were analyzed. Recovery of IKs from inhibition induced by muscarinic stimulation was incomplete despite full PIP2 resynthesis. Recovery of IKs was completely suppressed under ATP-free conditions, but partially restored by the ATP analog AMP-PCP, providing evidence that depletion of intracellular ATP inhibits IKs independent of PIP2-depletion. Simultaneous patch-clamp and FRET measurements in cells co-expressing Ci-VSP and the PIP2-FRET sensor revealed a component of IKs inhibition directly related to dynamic PIP2-depletion. A second component of inhibition was independent of acute changes in PIP2 and could be mimicked by ATP-free pipette solution, suggesting that it results from intracellular ATP-depletion. The reduction of intracellular ATP upon Ci-VSP activation appears to be independent of its activity as a phosphoinositide phosphatase. Our data demonstrate that ATP-depletion slowed IKs activation but had no short-term effect on PIP2 regeneration, suggesting that impaired PIP2-resynthesis cannot account for the rapid IKs inhibition by ATP-depletion. Furthermore, the second component of IKs inhibition by Ci-VSP was reduced by AMP-PCP in the pipette filling solution, indicating that direct binding of ATP to the KCNQ1/KCNE1 complex is required for voltage activation of IKs. We suggest that fluctuations of the cellular metabolic state regulate IKs in parallel with Gq-coupled PLC activation and PIP2-depletion.

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. Early after the onset of hypertension, at 3 months of age, there were no differences in Ca2+ transients (CaTs) or expression and phosphorylation of Ca2+ handling proteins between SHR and WKY. At 7 months of age, when left ventricular (LV) hypertrophy had progressed and markers of fibrosis were increased in left atrium, CaTs (at 1 Hz stimulation) were still unchanged. Subcellular alterations in Ca2+ handling were observed, however, in SHR atrial myocytes including (i) reduced expression of the α1C subunit of and reduced Ca2+ influx through L-type Ca2+ channels, (ii) reduced expression of ryanodine receptors with increased phosphorylation at Ser2808, (iii) decreased activity of the Na+ / Ca2+ exchanger (at unaltered intracellular Na+ concentration), and (iv) increased SR Ca2+ load with reduced fractional release. These changes were associated with an increased propensity of SHR atrial myocytes to develop frequency-dependent, arrhythmogenic Ca2+ alternans. CONCLUSIONS: In SHR, hypertension induces early subcellular LA myocyte Ca2+ remodelling during compensated LV hypertrophy. In basal conditions, atrial myocyte CaTs are not changed. At increased stimulation frequency, however, SHR atrial myocytes become more prone to arrhythmogenic Ca2+ alternans, suggesting a link between hypertension, atrial Ca2+ homeostasis, and development of atrial tachyarrhythmias.

Differential effects of genetically-encoded Gßγ scavengers on receptor-activated and basal Kir3.1/Kir3.4 channel current in rat atrial myocytes.

Opening of G-protein-activated inward-rectifying K(+) (GIRK, Kir3) channels is regulated by interaction with ßγ-subunits of Pertussis-toxin-sensitive G proteins upon activation of appropriate GPCRs. In atrial and neuronal cells agonist-independent activity (I(basal)) contributes to the background K(+) conductance, important for stabilizing resting potential. Data obtained from the Kir3 signaling pathway reconstituted in Xenopus oocytes suggest that I(basal) requires free G(ßγ). In cells with intrinsic expression of Kir3 channels this issue has been scarcely addressed experimentally. Two G(ßγ)-binding proteins (myristoylated phosducin - mPhos - and G(αi1)) were expressed in atrial myocytes using adenoviral gene transfer, to interrupt G(ßγ)-signaling. Agonist-induced and basal currents were recorded using whole cell voltage-clamp. Expression of mPhos and G(αi1) reduced activation of Kir3 current via muscarinic M(2) receptors (IK(ACh)). Inhibition of IK(ACh) by mPhos consisted of an irreversible component and an agonist-dependent reversible component. Reduction in density of IK(ACh) by overexpressed Gαi1, in contrast to mPhos, was paralleled by substantial slowing of activation, suggesting a reduction in density of functional M2 receptors, rather than G(ßγ)-scavenging as underlying mechanism. In line with this notion, current density and activation kinetics were rescued by fusing the αi1-subunit to an Adenosine A(1) receptor. Neither mPhos nor G(αi1) had a significant effect on I(basal), defined by the inhibitory peptide tertiapin-Q. These data demonstrate that basal Kir3 current in a native environment is unrelated to G-protein signaling or agonist-independent free G(ßγ). Moreover, our results illustrate the importance of physiological expression levels of the signaling components in shaping key parameters of the response to an agonist.

Voltage-dependent open-channel block of G protein-gated inward-rectifying K(+) (GIRK) current in rat atrial myocytes by tamoxifen.

Tamoxifen (Tmx) is a nonsteroidal selective estrogen receptor antagonist and is frequently used in the treatment and prevention of breast cancer. The compound and its metabolites have been reported to inhibit functions of different classes of membrane proteins, including various ion channels. For members of the inward-rectifying K(+) (Kir) channel family, interference of Tmx with binding of phosphatidylinositol 4,5-bisphosphate (PIP(2)) has been suggested as the mechanism underlying such inhibition. We have studied the inhibition of G protein-activated K(+) (GIRK) current by Tmx in isolated myocytes from hearts of adult rats using whole-cell voltage clamp and experimental conditions for measuring K(+) currents as inward currents (E (K) -50 mV; holding potential -90 mV). Extracellular Tmx reversibly inhibited GIRK current activated by acetylcholine (I (K(ACh))) with an EC(50) of 7.4 × 10(-7) M. This inhibition was composed of two components, a basal reduction in peak current and a block that required opening of channels by ACh. The open-channel block was partially relieved by depolarizing voltage steps in a voltage- and time-dependent fashion. A voltage-dependent open-channel block was not observed when I (K(ACh)) was measured as outward current (E (K) -90 mV; holding potential -40 mV). Intracellular application of Tmx via the patch clamp pipette at a concentration (7 × 10(-6) M) that caused a rapid inhibition of I (K(ACh)) upon extracellular application did not affect the current. Intracellular application of the H(2)O-soluble PIP(2) analog diC(8)-PIP(2) reduced the voltage-independent component of inhibition but had no effect on voltage-dependent open-channel block. The effects of 4-hydroxy-Tmx, a major active metabolite, tested at 2 × 10(-6) M, had effects on I (K(ACh)) analogous to those of Tmx. Inhibition of constitutive inward-rectifying K(+) current (I (K1)) in ventricular myocytes, carried by Kir2 complexes, by Tmx was devoid of a voltage-dependent component. This study suggests the voltage-dependent open-channel block of GIRK inward current as a novel mechanism of Tmx action.

Altered stress stimulation of inward rectifier potassium channels in Andersen-Tawil syndrome.

Inward rectifier potassium channels of the Kir2 subfamily are important determinants of the electrical activity of brain and muscle cells. Genetic mutations in Kir2.1 associate with Andersen-Tawil syndrome (ATS), a familial disorder leading to stress-triggered periodic paralysis and ventricular arrhythmia. To identify the molecular mechanisms of this stress trigger, we analyze Kir channel function and localization electrophysiologically and by time-resolved confocal microscopy. Furthermore, we employ a mathematical model of muscular membrane potential. We identify a novel corticoid signaling pathway that, when activated by glucocorticoids, leads to enrichment of Kir2 channels in the plasma membranes of mammalian cell lines and isolated cardiac and skeletal muscle cells. We further demonstrate that activation of this pathway can either partly restore (40% of cases) or further impair (20% of cases) the function of mutant ATS channels, depending on the particular Kir2.1 mutation. This means that glucocorticoid treatment might either alleviate or deteriorate symptoms of ATS depending on the patient's individual Kir2.1 genotype. Thus, our findings provide a possible explanation for the contradictory effects of glucocorticoid treatment on symptoms in patients with ATS and may open new pathways for the design of personalized medicines in ATS therapy.

A genetically encoded tool kit for manipulating and monitoring membrane phosphatidylinositol 4,5-bisphosphate in intact cells.

BACKGROUND: Most ion channels are regulated by phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P(2)) in the cell membrane by diverse mechanisms. Important molecular tools to study ion channel regulation by PtdIns(4,5)P(2) in living cells have been developed in the past. These include fluorescent PH-domains as sensors for Förster resonance energy transfer (FRET), to monitor changes in plasma membrane(.) For controlled and reversible depletion of PtdIns(4,5)P(2), voltage-sensing phosphoinositide phosphatases (VSD) have been demonstrated as a superior tool, since they are independent of cellular signaling pathways. Combining these methods in intact cells requires multiple transfections. We used self-cleaving viral 2A-peptide sequences for adenovirus driven expression of the PH-domain of phospholipase-Cδ1 (PLCδ1) fused to ECFP and EYFP respectively and Ciona intestinalis VSP (Ci-VSP), from a single open reading frame (ORF) in adult rat cardiac myocytes. METHODS AND RESULTS: Expression and correct targeting of ECFP-PH-PLCδ1(,) EYFP-PH-PLCδ1, and Ci-VSP from a single tricistronic vector containing 2A-peptide sequences first was demonstrated in HEK293 cells by voltage-controlled FRET measurements and Western blotting. Adult rat cardiac myocytes expressed Ci-VSP and the two fluorescent PH-domains within 4 days after gene transfer using the vector integrated into an adenoviral construct. Activation of Ci-VSP by depolarization resulted in rapid changes in FRET ratio indicating depletion of PtdIns(4,5)P(2) in the plasma membrane. This was paralleled by inhibition of endogenous G protein activated K(+) (GIRK) current. By comparing changes in FRET and current, a component of GIRK inhibition by adrenergic receptors unrelated to depletion of PtdIns(4,5)P(2) was identified. CONCLUSIONS: Expression of a FRET sensor pair and Ci-VSP from a single ORF provides a useful approach to study regulation of ion channels by phosphoinositides in cell lines and transfection-resistant postmitotic cells. Generally, adenoviral constructs containing self-cleaving 2A-peptide sequences are highly suited for simultaneous transfer of multiple genes in adult cardiac myocytes.

Autocrine signaling via A(1) adenosine receptors causes downregulation of M(2) receptors in adult rat atrial myocytes in vitro.

G protein-activated K(+) channels composed of Kir3 (GIRK) subunits contribute to regulation of heart rate and excitability. Opening of these channels in myocytes is increased by binding of G(ßγ) upon activation of muscarinic M(2) receptors (M(2)-R) or A(1) adenosine receptors (A(1)-R). It has been shown that saturating activation of A(1)-R resulted in a smaller GIRK current than activation of M(2)-R. Adenovirus-driven overexpression of the A(1)-R caused an increase in current induced by adenosine (I(K(Ado))), whereas the M(2)-R-activated current (I(K(ACh))) was reduced. Here, we sought to get deeper insight into the mechanism causing this negative crosstalk. GIRK current in cultured rat atrial myocytes was recorded in whole cell mode. Adenovirus-driven RNA interference targeting the M(2)-R resulted in a reduction in I(K(ACh)) without affecting I(K(Ado)), arguing against a competition of the two receptors for common signaling complexes. The negative effect of A(1)-R overexpression on I(K(ACh)) was reduced by the A(1)-R antagonist DPCPX and augmented by the agonist chloro-N6-cyclopentyladenosin (CCPA). In native myocytes incubation with either CCPA or the muscarinic agonist carbachol resulted in reduction in I(K(ACh)) and I(K(Ado)), suggesting common pathways of A(1)-R and M(2)-R downregulation. In the absence of agonist, inhibition of adenosine deaminase by EHNA or exposure to AMP, less to ADP, but not ATP resulted in reduction of I(K(ACh)) and I(K(Ado)). Our data indicate that atrial myocytes generate adenosine from extracellular AMP, which activates A(1)-R in an autocrine fashion. Chronic activation of A(1)-R causes parallel downregulation of both A(1)-R and M(2)-R.

Pannexin 1 constitutes the large conductance cation channel of cardiac myocytes.

A large conductance (∼300 picosiemens) channel (LCC) of unknown molecular identity, activated by Ca(2+) release from the sarcoplasmic reticulum, particularly when augmented by caffeine, has been described previously in isolated cardiac myocytes. A potential candidate for this channel is pannexin 1 (Panx1), which has been shown to form large ion channels when expressed in Xenopus oocytes and mammalian cells. Panx1 function is implicated in ATP-mediated auto-/paracrine signaling, and a crucial role in several cell death pathways has been suggested. Here, we demonstrate that after culturing for 4 days LCC activity is no longer detected in myocytes but can be rescued by adenoviral gene transfer of Panx1. Endogenous LCCs and those related to expression of Panx1 share key pharmacological properties previously used for identifying and characterizing Panx1 channels. These data demonstrate that Panx1 constitutes the LCC of cardiac myocytes. Sporadic openings of single Panx1 channels in the absence of Ca(2+) release can trigger action potentials, suggesting that Panx1 channels potentially promote arrhythmogenic activities.

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. Incorporation of siRNA into terminally differentiated mammalian cells, such as adult cardiac myocytes is limited by their resistance to standard transfection protocols. Viral delivery of short-hairpin RNA (shRNA) overcomes these limitations and allows efficient gene silencing in these cells. This chapter describes the generation and characterization of recombinant siRNA-encoding adenoviruses and their application to adult cardiac myocytes, which represent a standard experimental model in research related to cardiac physiology and pathophysiology. Feasibility of this approach is demonstrated by effective ablation (>80%) of both, a transgene encoding for eGFP and the endogenous muscarinic M(2) acetylcholine receptor.

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). On the other hand in AM a large G protein gated current carried by Kir3.1/3.4 complexes can be activated by stimulation of muscarinic M(2) receptors (I(K(ACh))), which is assumed to be marginal in VM. Recent evidence suggests that total current carried by cardiac inward-rectifiers (I(K(ATP)), I(K(ACh)), I(K1)) in both, AM and VM is limited, due to K+ accumulation/depletion. This lead us to hypothesize that in conventional whole celI recordings I(K(ACh)) in VM is underestimated as a consequence of constitutive I(K1). In that case a reduction in density of I(K1) should be paralleled by an increase in density of I(K(ACh)). Three different experimental strategies have been used to test for this hypothesis: (i) Adenovirus-driven expression of a dominant-negative mutant of Kir2.1, one of the subunits supposed to form I(K1) channels, in VM caused a reduction in I(K1)-density by about 80 %. In those cells I(K(ACh)) was increased about 4 fold. (ii) A comparable increase in I(K(ACh)) was observed upon reduction of I(K1) caused by adenovirus-mediated RNA interference.(iii) Ba2+ in a concentration of 2 microM blocks I(K1) in VM by about 60 % without affecting atrial I(K(ACh)). The reduction in I(K1) by 2 microM Ba2+ is paralleled by a reversible increase in I(K(ACh)) by about 100%. These data demonstrate that the increase in K+ conductance underlying ventricular I(K(ACh)) is largely underestimated, suggesting that it might be of greater physiological relevance than previously thought.

A role for RGS10 in beta-adrenergic modulation of G-protein-activated K+ (GIRK) channel current in rat atrial myocytes.

The effect of beta-adrenergic stimulation on endogenous G-protein-activated K(+) (GIRK) current has been investigated in atrial myocytes from hearts of adult rats. Beta-adrenergic stimulation (10 microm isoprenaline, Iso) had no effect on activation kinetics, peak current or steady-state current but resulted in slowing of deactivation upon washout of acetylcholine (ACh), the time constant (tau(d)) being increased by a factor of about 2.5. The effect of Iso could be mimicked by inclusion of cAMP (500 microm) in the filling solution of the patch clamp pipette. The Iso-induced increase in tau(d) was blocked by the selective beta(1) receptor antagonist CGP-20112A (2 microm) and by the PKA inhibitor H9 (100 microm included in the pipette solution). A candidate for mediating these effects is RGS10, one of the regulators of G-protein signalling (RGS) species expressed in cardiac myocytes. Overexpression of RGS10 by adenoviral gene transfer resulted in a reduction in tau(d) of 60%. Sensitivity of tau(d) to Iso remained in cells overexpressing RGS10. Overexpression of RGS4 caused a comparable reduction in tau(d), which became insensitive to Iso. Expression of an RGS10 carrying a mutation (RGS10-S168A), which deletes a PKA phosphorylation site, caused a decrease in tau(d) comparable to overexpression of wild-type RGS10. Sensitivity of tau(d) to Iso was lost in RGS10-S168A-expressing myocytes. Silencing of RGS10 by means of adenovirus-mediated transcription of a short hairpin RNA did not affect basal tau(d) but removed sensitivity to Iso. These data suggest that endogenous RGS10 has GTPase-activating protein (GAP) activity on the G-protein species that mediates activation of atrial GIRK channels. Moreover, RGS10, via PKA-dependent phosphorylation, enables a crosstalk between beta-adrenergic and muscarinic cholinergic signalling.