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.

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.

Membrane Potential Controls the Efficacy of Catecholamine-induced ß1-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 ß1-adrenoreceptor (ß1-AR) by the catecholamines isoprenaline (Iso) and adrenaline (Adr) is regulated by V(M). This voltage-dependence is also transmitted to G protein and arrestin 3 signaling. Voltage-dependence of ß2-AR activation, however, was weak compared with ß1-AR voltage-dependence. Drug efficacy is a major target of ß1-AR voltage-dependence as depolarization attenuated receptor activation, even under saturating concentrations of agonists, with significantly faster kinetics than the deactivation upon agonist withdrawal. Also the efficacy of the endogenous full agonist adrenaline was reduced by depolarization. This is a unique finding since reports of natural full agonists at other voltage-dependent GPCRs only show alterations in affinity during depolarization. Based on a Boltzmann function fit to the relationship of V(M) and receptor-arrestin 3 interaction we determined the voltage-dependence with highest sensitivity in the physiological range of membrane potential. Our data suggest that under physiological conditions voltage regulates the activity of agonist-occupied ß1-adrenoceptors on a very fast time scale.

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 ß-arrestin binding, activation of G proteins, and G protein-activated inwardly rectifying K(+) currents. Comparison of the dynamics of voltage-dependence of clonidine- vs. norepinephrine-activated receptors uncovers interesting mechanistic insights. For norepinephrine, the time course of voltage-dependent deactivation reflected the deactivation kinetics of the receptor after agonist withdrawal and was strongly attenuated at saturating concentrations. In contrast, clonidine-activated α(2A)AR were switched by voltage even under fully saturating concentrations, and the kinetics of this switch was notably faster than dissociation of clonidine from α(2A)AR, indicating voltage-dependent regulation of the efficacy. We conclude that adrenergic receptors exhibit a unique, agonist-dependent mechanism of voltage-sensitivity that modulates downstream receptor signaling.

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. Upon activation of histaminergic H1 or muscarinic M3 receptors, a robust FRET signal occurred that allowed for the first time the analysis of the kinetics of this interaction in detail. On- and off-set kinetics of Gαq-p63RhoGEF interactions closely resembled the kinetics of Gαq activity. Analysis of the effect of RGS2 (regulator of G-protein signalling 2) on the dynamics of Gαq activity and their interaction with p63RhoGEF showed that RGS2 is able to accelerate both deactivation of Gαq proteins and dissociation of Gαq and p63RhoGEF to a similar extent. Furthermore, we were able to detect activation-dependent FRET between RGS2 and p63RhoGEF and observed a reduced p63RhoGEF-mediated downstream signalling in the presence of RGS2. In summary, these observations support the concept of a functional activation-dependent p63RhoGEF-Gαq-RGS2 complex.

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. A key event in this signalling system is the direct and dynamic interaction of Gαi1 subunits with adenylate cyclases. We established a FRET-based assay between G-protein subunits and AC5 (type 5 adenylate cyclase) and monitored receptor-stimulated interactions between Gαi1 and AC5 in single intact cells with high temporal resolution. We observed that FRET between Gαi1 and AC5 developed at much lower concentration of agonist compared with the overall Gi-protein activity resulting in a left-shift of the concentration-response curve by approximately one order of magnitude. Furthermore, Gi1-protein-mediated attenuation of AC5-dependent increases in cAMP occurred at comparable low concentrations of agonist. On analysing the dynamics we found the dissociation of the Gαi1 subunits and AC5 to occur significantly slower than the G-protein deactivation and to be insensitive to RGS4 (regulator of G-protein signalling type 4) expression. This led us to the conclusion that AC5, by binding active Gαi1, interferes with G-protein deactivation and reassembly and thereby might sensitize its own regulation.

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. This new tool allows continuous monitoring of transcriptional activity of NFAT in a physiological context in living cells. Furthermore, NFAT-RFP can be used simultaneously with NFAT-GFP fusion proteins to monitor transcriptional activity and subcellular localization of NFAT in the same cell.

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. Therefore, we tested the hypothesis whether IP(3) receptor activation regulates NFAT activity in cardiac myocytes. We used confocal microscopy to quantify the nuclear localization of NFATc1-green fluorescent protein (GFP) and NFATc3-GFP fusion proteins (quantified as the ratio of nuclear NFAT to cytoplasmic NFAT) in response to stimulation with neurohumoral agonists. In rabbit atrial myocytes, an overnight stimulation with endothelin-1, angiotensin II, and phenylephrine induced nuclear accumulation of NFATc1 that was sensitive to calcineurin inhibitors (cyclosporin A or inhibitor of NFAT-calcineurin association-6) and prevented by the IP(3) receptor inhibitor 2-aminoethoxydiphenyl borate. Furthermore, a direct elevation of intracellular IP(3) with a cell-permeable IP(3) acetoxymethyl ester (10 µM) induced nuclear localization of NFATc1. With a fluorescence-based in vivo assay, we showed that endothelin-1 also enhanced the transcriptional activity of NFATc1 in atrial cells. The agonists failed to activate NFATc1 in rabbit ventricular cells, which express IP(3) receptors at a lower density than atrial cells. They also did not activate NFATc3, an isoform that is highly influenced by nuclear export processes, in both cell types. Our data show that the second messenger IP(3) is directly involved in the activation of NFATc1 in adult atrial cardiomyocytes.

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. The activation and regulation of NFAT in adult cardiac myocytes, which may depend on the NFAT isoform and cell type, are not fully understood. This study compared basal localization, import, and export of NFATc1 and NFATc3 in adult atrial and ventricular myocytes to identify isoform- and tissue-specific regulatory mechanisms of NFAT activation under physiological conditions and in HF. NFAT-green fluorescent protein fusion proteins and NFAT immunocytochemistry were used to analyze NFAT regulation in adult cat and rabbit myocytes. NFATc1 displayed basal nuclear localization in atrial and ventricular myocytes, an effect that was attenuated by reducing intracellular Ca(2+) concentration and inhibiting calcineurin, and enhanced by the inhibition of nuclear export. In contrast, NFATc3 was localized to the cytoplasm but could be driven to the nucleus by angiotensin II and endothelin-1 stimulation in atrial, but not ventricular, cells. Inhibition of nuclear export (by leptomycin B) facilitated nuclear localization in both cell types. Ventricular myocytes from HF rabbits showed increased basal nuclear localization of endogenous NFATc3 and reduced responsiveness of NFAT translocation to phenylephrine stimulation. In control myocytes, Ca(2+) overload, leading to spontaneous Ca(2+) waves, induced substantial translocation of NFATc3 to the nucleus. We conclude that the activation of NFAT in adult cardiomyocytes is isoform and tissue specific and is tightly controlled by nuclear export. NFAT is activated in myocytes from HF animals and may be secondary to Ca(2+) overload.

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). The vasoactive agonists ATP (5 microM) or bradykinin (20 microM) in the presence of 2 mM extracellular Ca(2+) induced Ca(2+) release from the endoplasmic reticulum (ER) and activated capacitative Ca(2+) entry (CCE), which caused robust translocation of NFAT to the nucleus. This effect was sensitive to the CaN-inhibitor cyclosporin A (1 microM). Influx of extracellular Ca(2+) via CCE, but not ER Ca(2+) release was identified as the activating Ca(2+) source. NFAT was also activated by Ca(2+) influx induced by cell swelling, reverse mode Na/Ca exchange or ionomycin treatment. NFAT regulation was isoform-specific. Whereas activation of NFATc1-GFP by ATP resulted in persistent nuclear localization, NFATc3-GFP was only transiently imported into the nucleus, followed by rapid export back to the cytoplasm. Inhibition of nuclear kinases, which mediate export of NFAT via phosphorylation, or direct block of nuclear export (Leptomycin B) resulted in stable nuclear localization of NFATc3. These data demonstrate that extracellular Ca(2+) entry mediates NFAT activation. Furthermore, the regulation of nuclear localization of NFAT is isoform-specific and dependent on nuclear export processes.

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.

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.

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.

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. Here, we compared the modulation of M1-Rs and M3-Rs induced by changes in voltage to their allosteric modulation by chemical compounds. We quantified changes in receptor signaling in single HEK 293 cells with a FRET biosensor for the Gq protein cycle. In the presence of ACh, M1-R signaling was potentiated by voltage, similarly to positive allosteric modulation by benzyl quinolone carboxylic acid. Conversely, signaling of M3-R was attenuated by voltage or the negative allosteric modulator gallamine. Because the orthosteric site is highly conserved among M-Rs, but allosteric sites vary, we constructed "allosteric site" M3/M1-R chimeras and analyzed their voltage dependencies. Exchanging the entire allosteric sites eliminated the voltage sensitivity of ACh responses for both receptors, but did not affect their modulation by allosteric compounds. Furthermore, a point mutation in M3-Rs caused functional uncoupling of the allosteric and orthosteric sites and abolished voltage dependence. Molecular dynamics simulations of the receptor variants indicated a subtype-specific crosstalk between both sites, involving the conserved tyrosine lid structure of the orthosteric site. This molecular crosstalk leads to receptor subtype-specific voltage effects.

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. When human embryonic kidney (HEK) 293 cells expressing the individual receptors were stimulated with the agonist carbachol, membrane depolarization enhanced signaling by the M1 receptor but attenuated signaling by the M3 and M5 receptors. Furthermore, whether membrane depolarization enhanced or inhibited receptor signaling depended on the type of agonist. Membrane depolarization attenuated M3 receptor signaling when the receptor was bound to carbachol or acetylcholine, whereas depolarization enhanced signaling when the receptor was bound to either choline or pilocarpine. Docking calculations predicted that there were two distinct binding modes for these ligands, which were associated with the effect of depolarization on receptor function. From these calculations, we identified a residue in the M3 receptor that, when mutated, would alter the binding mode of carbachol to resemble that of pilocarpine in silico. Introduction of this mutated M3 receptor into cells confirmed that the membrane depolarization enhanced, rather than attenuated, signaling by the carbachol-bound receptor. Together, these data suggest that the directionality of the voltage sensitivity of GPCR signaling is defined by the specific binding mode of each ligand to the receptor.

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.

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 Kir3.1/Kir3.4 channels in mammalian atrial myocytes. We therefore studied how loading of adult atrial myocytes from rat hearts via the patch pipette filling solution with different concentrations of Na+ ([Na+]pip) affects Kir3 current. Surprisingly, in a range between 0 and 60 mm, Na+ neither had an effect on basal inward-rectifier current nor on the current activated by acetylcholine. Overexpression of Kir3.4 in adult atrial myocytes forced by adenoviral gene transfer results in formation of functional homomeric channels that interact with betagamma subunits upon activation of endogenous muscarinic receptors. These channels are activated at [Na+]pip >or= 15 mm, resulting in a receptor-independent basal inward rectifier current (I bir). I bir was neither affected by pertussis toxin nor by GDP-beta-S, suggesting G-protein-independent activation. PIP(2) depletion via endogenous PLC-coupled alpha1 adrenergic receptors causes inhibition of endogenous Kir3.1/3.4 channel currents by about 75%. In contrast, inhibition of Na+-activated I bir amounts to < 20%. The effect of the Kir3 channel blocker tertiapin-Q can be described using an IC50 of 12 nm (endogenous I K(ACh)) and 0.61 nm (I bir). These data clearly identify I bir as a homotetrameric Kir3.4 channel current with novel properties of regulation and pharmacology. Ibir shares some properties with a basal current recently described in atrial myocytes from an animal model of atrial fibrillation (AF) and AF patients.

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 (Kir2.1) in ACM in vitro using adenovirus driven transcription of siRNA hairpins. EGFP fluorescence and density of background inward rectifier current (IK1) were reduced by > 90% within about 6-8 days after transformation with the corresponding virus. In Kir2.1-silenced myocytes resting membrane potential was significantly reduced. Survival of these cells in culture was compromised, presumably due to Ca2+ -overload caused by the depolarization. The sequence-specific knockdowns of EGFP and Kir2.1 were confirmed on the RNA level using real-time RT-PCR. In Kir2.1-silenced myocytes density of transient outward current, carried predominantly by Kv4.x subunits remained unaffected. This communication for the first time demonstrates proof of principle of efficient RNA interference using adenovirus-based vectors and demonstrates its large potential in phenotyping of ACM.

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. In ventricular myocytes, the background current-voltage relation of which is dominated by a large constitutive inward rectifier (I(K1)), the slope conductance of I(K(ATP)) was reduced at membrane potentials negative to E(K) (around -50 mV), resulting in an outwardly rectifying I-V relation. Overexpression of Kir2.1 by adenoviral gene transfer, a subunit contributing to I(K1) channels, in atrial myocytes resulted in a large I(K1)-like background current. The I-V relation of I(K(ATP)) in these cells showed a reduced slope conductance negative to E(K) similar to ventricular myocytes. In atrial myocytes with an increased background inward-rectifier current through Kir3.1/Kir3.4 channels (I(K(ACh))), irreversibly activated by intracellular loading with GTP-gamma-S, the I-V relation of I(K(ATP)) showed a reduced slope negative to E(K), as in ventricular myocytes and atrial myocytes overexpressing Kir2.1. It is concluded that the voltage dependencies of membrane currents are not only dependent on the molecular composition of the charge-carrying channel complexes but can be affected by the activity of other ion channel species. We suggest that the interference between inward I(K(ATP)) and other inward rectifier currents in cardiac myocytes reflects steady-state changes in K+ driving force due to inward K+ current.

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. Acute desensitization was also identified as a decrease in current amplitude and a blunting of the response to saturating [ACh] (20 micromol l(-1)) when the current had been partially activated by a low concentration of ACh or by stimulation of adenosine A1 receptors. A reduction in current analogous to acute desensitization was observed when ATP-dependent K+ current (I(K(ATP))) was activated either by mitochondrial uncoupling using 2,4-dinitrophenole (DNP) or by the channel opener rilmakalim. Adenovirus-driven overexpression of Kir2.1, a subunit of constitutively active inwardly rectifying K+ channels, resulted in a large Ba2+-sensitive background K+ current and a dramatic reduction of ACh-activated current. Adenovirus-driven overexpression of GIRK4 (Kir3.4) subunits resulted in an increased agonist-independent GIRK current paralleled by a reduction in I(K(ACh)) and removal of the desensitizing component. These data indicate that acute desensitization depends on K+ current flow, independent of the K+ channel species, suggesting that it reflects a reduction in electrochemical driving force rather than a bona fide signalling mechanism. This is supported by the observation that desensitization is paralleled by a significant negative shift in reversal potential of I(K(ACh)). Since the ACh-induced hyperpolarization shows comparable desensitization properties as I(K(ACh)), this novel current-dependent desensitization is a physiologically relevant process, shaping the time course of parasympathetic bradycardia.