Marie-Cécile Kienitz

Pannexin 1 Constitutes the Large Conductance Cation Channel of Cardiac Myocytes

A large conductance (∼300 picosiemens) channel (LCC) of unknown molecular identity, activated by Ca2+ 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 Ca2+ release can trigger action potentials, suggesting that Panx1 channels potentially promote arrhythmogenic activities.

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

The effect of β‐adrenergic stimulation on endogenous G‐protein‐activated K+ (GIRK) current has been investigated in atrial myocytes from hearts of adult rats. β‐Adrenergic stimulation (10 μm 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 (τd) being increased by a factor of about 2.5. The effect of Iso could be mimicked by inclusion of cAMP (500 μm) in the filling solution of the patch clamp pipette. The Iso‐induced increase in τd was blocked by the selective β1 receptor antagonist CGP‐20112A (2 μm) and by the PKA inhibitor H9 (100 μm 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 τd of 60%. Sensitivity of τd to Iso remained in cells overexpressing RGS10. Overexpression of RGS4 caused a comparable reduction in τd, which became insensitive to Iso. Expression of an RGS10 carrying a mutation (RGS10‐S168A), which deletes a PKA phosphorylation site, caused a decrease in τd comparable to overexpression of wild‐type RGS10. Sensitivity of τd 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 τd 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 β‐adrenergic and muscarinic cholinergic signalling.

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 M₂ 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 µM blocks I_K1 in VM by about 60 % without affecting atrial I_K(ACh). The reduction in I_K1 by 2 µM 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.

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 βγ 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 βγ subunits upon activation of endogenous muscarinic receptors. These channels are activated at [Na+]pip≥ 15 mm, resulting in a receptor‐independent basal inward rectifier current (Ibir). Ibir was neither affected by pertussis toxin nor by GDP‐β‐S, suggesting G‐protein‐independent activation. PIP2 depletion via endogenous PLC‐coupled α1 adrenergic receptors causes inhibition of endogenous Kir3.1/3.4 channel currents by about 75%. In contrast, inhibition of Na+‐activated Ibir amounts to < 20%. The effect of the Kir3 channel blocker tertiapin‐Q can be described using an IC50 of 12 nm (endogenous IK(ACh)) and 0.61 nm (Ibir). These data clearly identify Ibir 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.

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.

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 patients 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.—Seebohm, G., Strutz‐Seebohm, N., Ursu, O. N., Preisig‐Müller, R., Zuzarte, M., Hill, E. V., Kienitz, M.‐C., Bendahhou, S., Fauler, M., Tapken, D., Decher, N., Collins, A., Jurkat‐Rott, K., Steinmeyer, K., Lehmann‐Horn, F., Daut, J., Tavaré, J. M., Pott, L., Bloch,W., Lang, F. Altered stress stimulation of inward rectifier potassium channels in Andersen‐Tawil syndrome. FASEB J. 26, 513–522 (2012). www.fasebj.org

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)P2) in the cell membrane by diverse mechanisms. Important molecular tools to study ion channel regulation by PtdIns(4,5)P2 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)P2, 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)P2 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)P2 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.

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.

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(βγ)-activated channels.

Differential effects of genetically-encoded Gβγ 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 βγ-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(βγ). In cells with intrinsic expression of Kir3 channels this issue has been scarcely addressed experimentally. Two G(βγ)-binding proteins (myristoylated phosducin - mPhos - and G(αi1)) were expressed in atrial myocytes using adenoviral gene transfer, to interrupt G(βγ)-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(βγ)-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(βγ). Moreover, our results illustrate the importance of physiological expression levels of the signaling components in shaping key parameters of the response to an agonist.