DREADD technology reveals major impact of G_q signalling on cardiac electrophysiology

Abstract

Aims

Signalling via Gq-coupled receptors is of profound importance in many cardiac diseases such as hypertrophy and arrhythmia. Nevertheless, owing to their widespread expression and the inability to selectively stimulate such receptors in vivo, their relevance for cardiac function is not well understood. We here use DREADD technology to understand the role of Gq-coupled signalling in vivo in cardiac function.

Methods and results

We generated a novel transgenic mouse line that expresses a Gq-coupled DREADD (Dq) in striated muscle under the control of the muscle creatine kinase promotor. In vivo injection of the DREADD agonist clozapine-N-oxide (CNO) resulted in a dose-dependent, rapid mortality of the animals. In vivo electrocardiogram data revealed severe cardiac arrhythmias including lack of P waves, atrioventricular block, and ventricular tachycardia. Following Dq activation, electrophysiological malfunction of the heart could be recapitulated in the isolated heart ex vivo. Individual ventricular and atrial myocytes displayed a positive inotropic response and arrhythmogenic events in the absence of altered action potentials. Ventricular tissue sections revealed a strong co-localization of Dq with the principal cardiac connexin CX43. Western blot analysis with phosphor-specific antibodies revealed strong phosphorylation of a PKC-dependent CX43 phosphorylation site following CNO application in vivo.

Conclusion

Activation of Gq-coupled signalling has a major impact on impulse generation, impulse propagation, and coordinated impulse delivery in the heart. Thus, Gq-coupled signalling does not only modulate the myocytes’ Ca²⁺ handling but also directly alters the heart’s electrophysiological properties such as intercellular communication. This study greatly advances our understanding of the plethora of modulatory influences of Gq signalling on the heart in vivo.

1. Introduction

G protein mediated transmembrane and intracellular signalling pathways are among the most frequent mechanisms involved in physiological and pathological processes in the mammalian heart. Drugs that target G protein-coupled receptors (GPCRs) are amongst the most common prescriptions to treat cardiovascular diseases. However, there is an urgent need for further elucidation of the complex Gi-, Gs-, Go-, and Gq/11-signalling pathways as heart failure is still the top cause of death in developed countries.^1,2 Various knockout models for particular G-proteins have demonstrated their critical roles in cardiac development, morphology, and function.^3–6 However, it is difficult to study the function of specific GPCRs expressed in the heart by using selective GPCR agonists in vivo, primarily due to the fact that essentially all GPCRs are expressed in multiple tissues and cell types.

Optogenetic approaches involving the expression of light-sensitive receptors under cell-type specific promoters enabled light-induced G-protein activation in cardiomyocytes.⁷ However, light needs to be very close to the target tissue or organ and in vivo investigations often need to be performed in open-chest approaches. The application of optogenetic technology in unaffected, awake and freely moving animals is therefore rather challenging.

DREADD (designer receptors solely activated by designer drugs) technology overcomes these limitations by using a combined genetic/chemical approach.^8–12 These genetically modified designer GPCRs are unable to bind endogenous ligands, but can be activated by otherwise inert small molecules.¹³ The most commonly used DREADDs represent mutant muscarinic receptors that can be specifically activated by the DREADD agonist clozapine-N-oxide (CNO) but not by their natural ligand acetylcholine.¹⁴ DREADDs show only minimal constitutive activity even at high levels of expression and no noteworthy response to acetylcholine,^14,15 Up to now, DREADD technology has been mostly used by neuroscientists, but DREADDs have also been expressed in pancreatic β-cells^15,16, hepatocytes,¹² and cancer cells.¹⁷

Here, we present the first transgenic mouse line expressing a functional Gq-coupled designer receptor, M3Dq (Dq)¹⁵ under the transcriptional control of the muscle creatine kinase (MCK) promoter. We found that Dq expression was restricted to striated muscle. Basic characterization of the mice revealed the lack of a phenotype in the absence of CNO. However, CNO-mediated activation of Dq in vivo resulted in profound and specific alterations of the heart’s electrophysiological properties, highlighting the relevance of Gq signalling for the heart’s physiology and pathophysiology on the cellular, organ and systemic level.

2. Methods

2.1 Animal care

All animal procedures were approved by the Saarland State Office for Health and Consumer Protection (Permit Number: 41/2012). All animal experiments conform to the guidelines from Directive 2010/63/EU of the European Parliament on the protection of animals used for scientific purposes and the NIH guidelines (Guide for the care and use of laboratory animals). Mice were kept in a 12-h light/12-h dark cycle with ad libitum access to water and food.

2.2 Mouse line

MCK-Dq mice were generated by DNA microinjection performed by the NIDDK Mouse Transgenic Core facility. Briefly, the RIPII promoter from the RIPII-Rq construct¹⁵ was replaced with the MCK promoter segment excised from the pBS-MCK plasmid (Addgene, USA, #12528). Following confirmation of successful ligation, a 10.4 kb fragment containing the MCK promoter, the Gq DREADD sequence (Dq), and 3′-untranslated human growth hormone sequence was linearized with EcoRV and EagI and purified by agarose gel electrophoresis (Qiagen, USA). The purified 10.4 kb fragment was microinjected into the pronuclei of ova prepared from C57BL/6 mice (Taconic). Transgenic founder mice were identified by PCR analysis of mouse tail DNA. Transgenic mice were maintained on a pure C57BL/6 background. Mice heterozygous for the MCK-Dq transgene were mated with C57Bl6/6J mice (Charles River, Germany) to obtain heterozygous offspring and wild-type (WT) littermate controls.

2.3 Genotyping of mice

Genotyping of MCK-Dq mice was performed by PCR. A 147 bp PCR product was amplified using the following transgene-specific primers: 5´-CCGACTACGCCACCTT (forward), 5´-CAGTTTCTTGTGAAATGTTGTAGCTGC (reverse). The DreamTaq DNA Polymerase (ThermoFisher Scientific, Germany) was used. PCR cycling conditions were: 95°C for 2 min, followed by 35 cycles at 95°C for 30 s, 57°C for 30 s, and 72°C for 60 s.

2.4 Echocardiography

Cardiac ultrasonographic analysis was performed with the Vevo 770 system (Visualsonics, Canada) using a 30 MHz transducer, and anaesthesia with isoflurane (3.5% introduction, 1.5% maintenance, in O₂). The fur of the chest was removed, and the mouse was placed on a heated table with electrocardiogram (ECG) electrodes. An additional rectal probe was employed to monitor the body temperature. In B-mode, the parasternal long- and short-axis views were adjusted, and then M-mode-traces were recorded for each position to measure the morphological parameters of the heart. The blood flow velocity profile through the mitral valve was assessed in the four-chamber view, E-/A-wave velocities and the isovolumetric relaxation time were measured. With determination of the basic morphological parameters the ventricular volume in systole and diastole, the ejection fraction, fractional shortening, and the left ventricular mass were calculated on the system. This mass was then normalized to the body weight of the mouse.

2.5 Gene expression analysis

Ventricles of WT and MCK-Dq mice were frozen in liquid nitrogen and stored at −80° C until RNA extraction. Total RNA was prepared using the ISOLATE II RNA MiniKit (Bioline, Germany). RNA was eluted with 30 µL RNase-free water. The concentration and the quality of the RNA samples were determined in a UV spectrophotometer by measuring the absorption of light at 260 nm and 280 nm using a TrayCell cuvette (Hellma, Germany). cDNA was produced using the SensiFAST™ cDNA Synthesis Kit (Bioline, Germany) with a mixture of random hexamer and oligo(dT) primers. cDNA synthesis was performed with 10 ng of total RNA. The resulting cDNA was diluted to 0.5 ng/µL prior to use in qPCR that was performed with a MX3000P (Stratagene, USA) in 96-well microtiter plates using a final volume of 20 µL. Reaction mix was prepared with the SensiFAST SYBR Lo-ROX Kit (Bioline, Germany) which contains all the components necessary for real-time PCR, including the SYBR^® Green I dye, dNTPs, stabilizers and enhancers. Finally, 5 µL of template cDNA (2.5 ng) were added. Amplifications were started with a 10-min initial denaturation step at 95°C, followed by 40 cycles of denaturation at 95°C for 5 s, primer annealing at the optimized primer temperature for 10 s and a 1 min extension step at 72°C. After 40 cycles, a dissociation curve was performed for quality control. Real-time PCR was performed in triplicate for all genes in each sample and C_T values were calculated after an automated correction step in Igor Pro (WaveMatrix, USA) taking into account different PCR efficiencies of the primers and a specific threshold for every single run. C_T values were then normalized to the transcription level of the transcription factor GATA-4 (Gata4). Sequences of gene specific primers can be found in Table 1. The normalized C_T values were tested for differences in the Dq expression level between transgenic and WT samples of littermates.

Table 1.

List of primers used for qPCR analysis

Gene	Sense primer	Antisense primer
Dq (r/hM3Dq)	CCCTACGACGTCC CCGACTACG	GACCTTAAATGAC CAATTACCA
Gata4	CTGAATAAATCTAA GACGCCAG	GACACAGTACTGA ATGTCTGT

2.6 Western blot analysis

For protein isolation frozen ventricle were homogenized using the MINILYS apparatus (Peqlab, Germany) and lysed with a buffer containing (in mM) Tris–HCl 100, NaCl 100, 0.5% Triton X-100, dithiothreitol 20, 143 µL/mL 7×Complete™ and 100 µL/mL 10×PhosSTOP™ (Roche Applied Science, Switzerland) at a pH of 7.5. Cell debris was removed by centrifugation (4°C, 30 min, 13 000×g). The protein content was determined according to standard Bradford test. Proteins were analysed by SDS-polyacrylamide gel electrophoresis. They were separated on a 12% gel (NuPAGE Bis/Tris gel, Invitrogen) following the instructions of the manufacturer. For western blot analysis proteins were transferred to a nitrocellulose membrane by tank blotting with NuPage transfer buffer (with 5%, v/v methanol). Membranes were blocked in 4% (v/v) blocking solution (Roti-Block, Carl Roth, Germany) for 1 h at room temperature. The membrane was incubated with the primary antibodies in PBS-Tween20 with 4% blocking solution over night at 4°C. We used the rabbit anti-Cx43-antibody (1:5000) and rabbit anti pS368-antibody (1:500) (Abcam, UK). The membrane was washed with PBS-Tween20 three times before incubating with the peroxidase-coupled secondary antibodies (Abcam, UK) in a dilution of 1:10 000 in PBS-Tween20 with 4% blocking solution. Signals were developed and visualized by the Lumilight system (Roche Applied Science, Switzerland). Signal strength was quantified following import of the digitized membranes into ImageJ.

2.7 Cardiomyocyte isolation

The preparation of isolated ventricular and atrial myocytes was described previously.^18,19 Ten- to 12-week-old mice were anaesthetized with a mixture of xylazine hydrochloride (85 mg/kg body weight, Rompun^® 20 mg/mL, Bayer, Germany) and ketamine hydrochloride (15 mg/kg body weight, Ursotamin^® 100 mg/mL, Serumwerk Bernburg, Germany) dissolved in isotonic saline. Sodium citrate (117 mg/kg body weight) was injected to prevent blood clotting. All administration routes were intraperitoneal (i.p.). In the absence of any withdrawal reflex, mice were killed by decapitation, the chest was opened and 2 mL of ice-cold solution A+ (in mM): NaCl 134, KCl 4, glucose 11, MgSO₄ 1.2, Na₂HPO₄ 1.2, HEPES 10, EGTA 0.2, pH 7.35 was injected into both ventricles. Afterwards, the heart was quickly removed and cannulated for Langendorff perfusion with Solution A+ for 5 min at room temperature, followed by the digestion step. Thereafter, 1 mL of a frozen Liberase TM Research Grade (Roche, Switzerland) stock (2 mg/mL aqua dest.) was diluted to a final concentration of 0.125 mg/mL with Solution A (in mM): NaCl 134, KCl 4, glucose 11, MgSO₄ 1.2, Na₂HPO₄ 1.2, HEPES 10, pH 7.35, and Langendorff perfused for 12 min at 37°C. The ventricles and atria were washed in solution A, then in oxygenated Solution A++ (37°C). Solution A++ comprised 0.1% DNAse [15 mg deoxyribonuclease I, type 2 from bovine pancreas (Sigma Aldrich, Germany)] and Solution A [10 mmol/L Tris HCl, 50 mmol/L NaCl, 10 mmol/L MgCl₂, 1 mmol/L DTT (pH 7.35) and glycerol]. The dissected ventricles or atria were placed in 5 mL fresh, oxygenated solution A++ and dissociated. The Ca²⁺ concentration was increased by repetitive addition of 150 µL Solution B (Solution A++) for 10 times in 5–8 min intervals. The cell suspension was diluted in medium [MEM, 1% (v/v) penicillin-streptomycin-glutamine 100×, 50 µg/L kanamycin A, 5 mg/L apo-transferrin human, 5 mg/L insulin from bovine pancreas, 29 nmol Na₂SeO₃] to yield the desired cell density and dispersed to ECM gel (Sigma-Aldrich, Germany) coated coverslips. Cells were incubated at 37°C with 5% CO₂. The isolation protocol was completed by changing the medium after 60 min.

2.8 Immunofluorescent labelling, confocal and super-resolution imaging

The hearts of anaesthetized mice were flushed from blood with 1×PBS (5 mL) by punctuating the right and left ventricles with a 26 gauge canula. Hearts were quickly removed, immediately perfused with 4% paraformaldehyde (in PBS) through the aorta for rapid fixation. In depth fixation was performed in 4% paraformaldehyde over night at 4°C. Hearts were then cryoprotected in 18% sucrose in PBS overnight and embedded in Optimal Cutting Temperature compound (OCT, Leica, USA). A glass container was placed in a mixture of ethanol and dry ice and then partially filled with isopentane. Hearts were placed in individual wells of a 12-well plate filled with OCT for 3–4 h, gently squeezing every 30 min to completely fill the ventricles with the embedding medium. Hearts were then mounted in plastic cuvettes filled with OCT, placed into the isopentane bath for rapid freezing and stored at −80°C. Embedded hearts were cut into 14 µm sections using a Leica cryostat (CM3000 equipped with a 2045C stage, Leica, Germany) and then probed with primary antibodies; rabbit anti-HA (Sigma, Germany) and mouse anti-BIN1 (Sigma), mouse anti-NCX (Jackson Immunoresearch, USA) and mouse anti-PMCA (Abcam, Germany). For these primary antibodies, we utilized anti-rabbit 594 and anti-mouse 649 as secondary antibodies (both Thermo Fisher, Germany). We combined the rabbit antiCx43 with the mouse anti-HA (BioLegend, Germany) and used anti-mouse 594 and anti-rabbit 649 as secondary antibodies (both Jackson Immunoresearch, USA). Labelled cryosections were mounted in mounting medium (Fluomount™ Aequous mounting medium, Sigma/Germany) and analysed with a Leica SP5-II confocal microscope (Leica Microsystems, Germany). Excitation of the 549 and 594 labels was performed with a 561 nm solid state laser while the 649 labels were excited with a 638 nm HeNe laser. The emission was collected with the spectral detector of the confocal unit. When combining the 549/649 probes, imaging of both labels was performed simultaneously (emission bands were: 565–630 nm and 640–750 nm, respectively). For the 594/633 combination, we performed imaging successively at 561 nm excitation (565–660 nm emission) followed by 638 nm excitation (640–750 nm emission) for each confocal imaging plane. Imaging was performed through a 63× oil immersion objective (Leica 63×, NA-1.40-HCX PL APO) with the image size set to 1024 × 1024 pixels resulting in pixel dimensions of 141 nm × 141 nm (in x and y). The pinhole was closed yielding confocal sections of around 770 nm thickness.

Super-resolution imaging was performed with an iSIM microscope (VisiTech Int., UK) using a 100× oil immersion objective (SR Apo TIRF, NA 1.49, NIKON, Japan). The super-resolution head was mounted to an inverted microscope (Eclpise Ti, NIKON, Japan) equipped with a motorized x/y table (Ludl, USA) and a piezo-controlled z-drive (Physik Instrumente, Germany). Excitation was achieved by two solid state lasers (561 nm and 638 nm, both Coherent, USA) and emission was collected through two emission bands (570–620 nm and >645 nm) and imaged with a sCMOS camera (Flash4, HAMAMTSU, Japan). Images (1516 × 1024 pixel in x/y direction) were collected and processed as described above. After deconvolution, the point spread function evaluated with 100 nm fluorescence beads yielded an optical resolution of around 120 nm × 120 nm × 290 nm (x, y, z direction).

All images were transferred into a large-scale image repository database running OMERO (Open microscopy environment, University of Dundee, UK) for long-term storage.^20,21 Further processing such as transforming the 12-bit grey-scale images into pseudo-coloured RGB images was performed in ImageJ software (W. Rasband, NIH, USA). For deconvolution of the confocal and super-resolution data we utilized AutoQuantX3 (MediaCybernetics, USA). Three-dimensional reconstruction (volume rendering) was performed with Imaris software (version 9.1; BitPlane, CH). Line profiles and Fourier transformations were calculated using ImageJ (W. Rasband, NIH, USA) and Igor (version 7.0; Wavemetrics, USA).

2.9 Cellular calcium transients

Cellular, global Ca²⁺ transients were monitored using Fura-2/AM loaded myocytes as previously described.^18,19 The coverslips with the Fura-2 loaded cardiomyocytes were mounted in a custom-made chamber on the stage of an inverted microscope (uiMic, TILL Photonics, Germany) attached to a video-imaging system. The system comprised a fully automated imaging stage, a CCD camera (Retiga 2000R, QImaging, Czech Republic), and a fast monochromator for excitation (Polychrome IV, TILL Photonics, Germany). The entire setup was controlled by an Integrated Control Unit (ICU, TILL Photonics, Germany) and driven by Live Acquisition software (TILL Photonics, Germany). Excitation was performed at alternating wavelengths 350/380 nm (7 nm FWHM). The emitted Fura-2 fluorescence was detected through a 20× oil-immersion objective (Planfluor 0.75 NA, Olympus, Germany), reflected to and recorded with the CCD camera (8 × 8 binning—resulting image size 200 × 150 pixels). Fura-2 ratios were obtained at 60 Hz.

Prior to an experiment, both ventricular and atrial cells were loaded with Fura-2/AM (1 μmol/L, from a stock of 1 mmol/L in DMSO/20% pluronic; Invitrogen, Darmstadt, Germany) for 30 min, followed by an additional 10 min de-esterification period in dye-free Tyrode's solution (in mmol/L: KCl 5.4, NaCl 135, MgCl₂ 1, HEPES 10, CaCl₂ 1.8, glucose 10, pH 7.35 adjusted with NaOH). During the entire imaging experiments, the cells were perfused with Tyrode by a local solenoid-controlled gravity-driven perfusion system. Electrical excitation of the cells was performed with two parallel platinum wires [10 mm apart; 15–20 V square alternating pulses at 2 Hz, MyoPacer (IonOptix, Ireland)]. Before performing the actual experiment, seven recording fields (so called ‘markers’ in the software) were randomly picked. For each field, a recording of 10 s was performed. On completion of recording, the fully automated stage moved to the next ‘marker position’ automatically. The whole imaging process from the first marker to the last marker lasted about 80 s. The same process was repeated for at least 15 times. All experiments were performed at 33–35°C.

The acquired imaging data were transferred into a large-scale image repository database running OMERO (Open microscopy environment, University of Dundee, UK) for long-term storage.^20,21 Analysis of the imaging data was performed with custom made macros in ImageJ (W. Rasband, NIH, Bethesda, USA), Igor (Wavemetrics, Lake Oswego, USA) and Matlab (Mathworks, USA). We extracted background-corrected fluorescence information and calculated Fura-2 ratios by dividing the integrated region-of-interest fluorescence of the 350 nm (excitation) image by the value obtained from the 380 nm image (excitation). From these ratio data, characteristic values (transient amplitude, baseline ratio, etc.) were calculated with custom procedures running in Igor or Matlab and exported into data files for later statistical analysis (see below).

2.10 Electrophysiology of action potentials

Action potentials were recorded at 34°C using a HEKA10 amplifier (HEKA Electronic, Germany). Patch pipettes had a resistance of 3–5 MΩ when filled with internal solution (mM): KCl 135, NaCl 10, EGTA 1, MgCl₂ 2, HEPES 10, MgATP 3, pH was adjusted to 7.35 with KOH. The bath solution (Tyrode) contained (mM): NaCl 137, KCl 5.4, HEPES 10, glucose 10, MgCl₂ 1, CaCl₂ 1.8, pH was adjusted to 7.4 with NaOH. A short pulse (700–900 pA, 4 ms) with a frequency of 2 Hz was used repeatedly to induce action potentials from the isolated mouse ventricular cardiac myocytes, which had been perfused with Tyrode for 4 min then followed by CNO (10 µM) perfusion for 6 min.

2.11 Langendorff/isolated heart measurements

Ten- to 12-week-old male mice were anaesthetized with a mixture of xylazine hydrochloride (85 mg/kg body weight, Rompun^® 20 mg/mL, Bayer, Germany) and ketamine hydrochloride (15 mg/kg body weight, Ursotamin^® 100 mg/mL, Serumwerk Bernburg, Germany) dissolved in isotonic saline (i.p. administration route). Mice were decapitated after the absence of a withdrawal reflex. The hearts were connected at their aortic stump to an 18 gauge, blunted cannula and mounted to a Langendorff perfusion system for isolated mouse hearts (Radnoti, Ireland) for retrograde perfusion with oxygenated Tyrode's solution (in mM: NaCl 135, KCl 5, 4, glucose 10, MgCl₂ 1, HEPES 10, CaCl₂ 1.8, pH 7.35, 37°C). The hearts were allowed to recover for a few minutes. Perfusion pressure was kept constant at 75 mm Hg. In the meantime, a spring loaded, platinum tip electrode was placed on the epicardial site of the heart’s apex, and a second electrode in form of a platinum wire was wrapped around the aortic cannula. These leads (Radnoti, Ireland) were positive and negative electrodes to gain surface ECG from isolated, spontaneous beating hearts. The signal was amplified (Animal Bioamp, ADInstruments, UK) and acquired with Poweramp 8/35 (ADInstruments, UK). The heart was surrounded by a water-jacketed, glass-made heart chamber to keep the environment and therefore the tissue constant at 37°C. Following these steps, the perfusion was switched to oxygenated Tyrode's solution containing 7.5 µM CNO. Real-time monitoring and post-recording analysis was performed with LabChart Pro 7 (ADInstruments, UK). To determine R-wave amplitude, PQ segment duration and QRS complex duration, the mean out of 4 consecutive ECGs was averaged within a 20 s segment. Measurement time points were 1–2 min before CNO application and 10 s, 1 min, 2 min, 3 min, and 5 min after CNO application.

2.12 Telemetric measurement of electrocardiogram and blood pressure

The ECG and mean arterial pressure were recorded by using a telemetric, implantable device (HD-X11, Data Sciences International, USA). For the implantation, 9–11-week old male MCK-Dq and WT littermate mice were anaesthetized (3% isoflurane for induction, 1.5% maintenance, analgesia: carprofen (10 mg/kg, Rimadyl^® 50 mg/ml, Zoetis, Germany); antibiosis: enrofloxacin (7.5 mg/kg, Baytril^®, Bayer, Germany)). The animal’s forelimbs were taped in dorsal recumbency to a heating plate after extinction of the withdrawal reflex. The shaved and disinfected chest was incised from the mandible to the sternum, the underlying salivary glands were gently separated, and the left carotid artery was carefully isolated with blunted, curved forceps from all surrounding tissues. Three 10 cm pieces of 6/0 silk suture were positioned under the artery. The furthest cranial suture was tightly ligated at the height of the bifurcation of the interior and exterior carotid arteries. A loose knot in the two other sutures and an additional micro serrefine temporarily occluded blood flow to prevent bleeding during cannulation and pressure catheter insertion. The carotid artery was pierced with a modified 26-gauge needle proximal to the permanent knot. The tip of the blood pressure catheter was inserted and advanced until the tip reached the aortic arch. Therefore, the serrefine had to be removed and the loose knots released further to allow passage of the catheter. Afterwards, the knots were permanently tightened to keep the catheter in position in the aortic arch. A subcutaneous pocket from the caudal end of the incision to the right limb was formed by blunt dissection for implantation of the device body. The XD-11’s negative lead was fixed subcutaneously to the right pectoral muscle, the positive lead via an additional subcutaneous tunnel 10 mm left to the manubrium sterni. The neck incision was closed and the animals were allowed to recover for 8 days (additional analgesic and antibiotic treatment on day 1 and day 2 after the implantation). Transgenic and WT mice underwent two measurements on two consecutive days, one for a isotonic saline control and one for CNO (1 or 10 mg/kg body weight CNO dissolved in isotonic saline) (i.p. injection). Measurements were performed at 30°C environmental temperature and started 30 min prior to injections. The endpoint of measurements was defined as the time point of death or maximum 12 h after injection. ECG and blood pressure data were collected with Dataquest A.R.T. Silver (Data Sciences International, USA) with a sampling rate of 500 Hz. ECG data analysis was performed with LabChart Pro 7 (ADInstruments, UK). To determine PQ interval duration, QRS complex duration and corrected QT segment duration (QTc = QT/√(RR/100), 1-min-sections were divided in six segments and further subdivided in 10 sub segments; the single ECG cycles within the sub segments were averaged. The mean of these values represented the parameters associated with a defined time point. Defined time points were 5 min before saline/CNO injection, minute-by-minute during the first 10 min, 20, 25, 30, 35, 40, 45, 50, 55, and 60 min after injection. The mean (systemic) arterial pressure (MAP) was calculated by averaging a 60 s segment at the same defined time points.

2.13 CNO plasma concentration

To determine the CNO plasma concentrations in vivo, we injected CNO (10 mg/kg i.p.) into WT mice and collected blood via cardiac puncture in heparinized tubes after 15 min. Samples were centrifuged immediately after (15 min, 2000 g) for plasma separation and frozen at −20°C. CNO concentrations were determined via HPLC-MS at Pharmacelsus GmbH, Germany.

2.14 Statistical analysis

Statistical analysis was performed in Prism 6&7 (GraphPad, USA). Normal distribution of the data was tested with the D’Agostino–Pearson omnibus normality test. In case of a normal distribution, the data were analysed with an unpaired t-test and displayed as bar graphs depicting mean ± SEM. The data in Figure 2 are displayed as a scatter plot and the mean ± standard deviation (SD). In the absence of a normal distribution, the data were analysed using a Mann–Whitney test. We displayed the data as box plots (box shows the median and the 25th to the 75th percentiles while the whiskers depict the 10th to the 90th percentiles). Statistical significance was defined as follows; P < 0.05 (*), P < 0.01 (**), P < 0.001 (***). N numbers are explicitly explained in the figure legends due to a mix of animals, organs, and cells.

Figure 2.

In vivo activation of Dq induces severe cardiac arrhythmias in Dq⁺ mice. (A) Basic experimental design and colour-coded occurrence of various types of cardiac arrhythmias (legend to the right). WT and Dq⁺ mice were injected with either saline or CNO (10 mg/kg i.p.). ECGs and the mean arterial pressure were measured by telemetry. Cardiac arrhythmias occurred only in CNO-treated Dq⁺ mice. Typical ECGs for the highlighted time points were replotted in B–E. (B) Representative control ECGs in a Dq⁺ mouse before CNO injection. Part of the tracing marked by the dashed box was replotted at an expanded time to the right. The PQ interval is marked in green. (C) Typical ECG recording 5 min after CNO injection depicting markedly altered ECGs. At an expanded timescale (right), the alterations became very clear. The prolonged PQ interval indicates AV block Type 1. (D) 15 min after CNO injection, the ECG tracings indicate that P-waves lack a following QRS complex (AV block Type II). (E) Forty-five minutes after CNO injection, we observed the occurrence of frequent intermittent ventricular tachycardia and AV dissociation. The mice often died when such arrhythmias occurred. Statistical analysis of (F) PQ interval, (G) QRS complex duration, and (H) QTc interval. (I) Time course of the mean arterial pressure recorded during the experiments. Each data point represents the mean of 7–9 mice. The colour code for (F–I) is indicated at the bottom of the figure. Black, dashed lines in (F–I) indicate significant differences between CNO-treated Dq⁺ mice vs. all other experimental groups. Data were analysed with an unpaired t-test, whereby each time point for all conditions was probed against the ‘WT Saline’ data. Data points depict the mean values ± SEM.

3. Results

3.1 Characterization of the novel MCK-Dq mouse line

qRT-PCR studies clearly demonstrated the expression of Dq in cardiac tissue of MCK-Dq positive mice (Dq⁺) but not in MCK-Dq negative littermates (WT) (Figure 1A). Consistent results were obtained using western blotting of cardiac samples from Dq⁺ mice (Figure 1B). For western blotting studies, we used an anti-HA antibody targeted against the HA-tag present at the N-terminus of Dq. Figure 1C shows that the Dq protein was selectively expressed in striated muscle.

Figure 1.

Analysis of Dq expression and its subcellular localization. (A) qPCR detection of the Dq transcript is specific to Dq⁺ mice (red), while WT mice lack such a signal (black). Statistical analysis was done with 16 WT and 22 Dq⁺ animals. After testing for normal distribution with a D’Agostino–Pearson omnibus normality test, the data were analysed with an unpaired t-test and the lines indicate the mean values ± SEM. (B) Western blot of three Dq⁺ (left lanes) and a WT mouse heart (rightmost lane) with loading control (GAPDH). (C) Western blot indicating the tissue-specific expression of Dq. Labels at the top of the individual lanes depict the source of the samples. Similar results were obtained with five additional mice. Dq was detected by using an antibody directed against the HA tag fused to the N-terminus of Dq. (D) Typical HA-positive (Da) and NCX (Db) immunofluorescence pattern in ventricular cryosections. The overlay of both signals is depicted in (Dc). Areas marked with a dashed box have been re-plotted at a higher magnification as insets. (Dd) Fluorescence profiles for Dq and NCX immunoreactivity along the yellow dashed lines in Da and Db is shown. Locations of positive immunoreactivity have been marked by green (Dq) and red (NCX) arrows. (De) Power-spectral analysis of the line profiles shown in (Dd) display the typical sarcomeric spatial frequency (black arrowhead). Same patterns were found in three Dq positive mouse hearts for which a total of six cryosection each were investigated. For background stainings, we refer to Supplementary material online, Figure S2.

We next probed Dq expression on the cellular level and its subcellular distribution in ventricular tissue (Figure 1D). In cryosections from WT mice, Dq immunofluorescence was virtually absent (Supplementary material online, Figure S2). In contrast, Dq⁺ ventricular tissue showed strong immunoreactivity with a clear cross striation pattern (Figure 1Da and inset). Co-localization analysis of the Dq immunoreactivity with the Na/Ca-exchanger (NCX, Figure 1D), the plasma membrane Ca-ATPase (PMCA, Supplementary material online, Figure S1A) and BIN1 (Supplementary material online, Figure S1B) displayed a pronounced overlap of these plasma membrane markers and similar spatial patterns (Figure 1De, Supplementary material online, Figures S1Ae and Be), strongly suggesting a plasma membrane localization of Dq.

In a cohort of Dq⁺ and WT male littermates, we investigated potential alterations of the heart’s morphology and function by echocardiography (Supplementary material online, Figure S3). Cardiac morphology and function were not significantly altered by the expression of Dq, consistent with previous findings that this DREADD lacks constitutive activity.¹⁵

3.2 Activation of Dq results in dose-dependent mortality

CNO-induced activation of Dq in MCK-Dq mice resulted in dose-dependent mortality (Supplementary material online, Figure S4). A relatively low dose of CNO (1 mg/kg i.p.) caused the death of two out of five mutant mice after 16 h (Supplementary material online, Figure S4A). An increase of the CNO dose to 10 mg/kg i.p. led to the death of all MCK-Dq mice after approximately 4 h (Supplementary material online, Figure S4B). CNO injections into WT mice or saline injection into Dq positive mice did not cause any deaths (Supplementary material online, Figure S4B). Fifteen minutes after i.p. injection of 10 mg/kg CNO, the CNO plasma concentration was 7.43 ± 0.66 µM (n = 5 animals).

3.3 In vivo Dq activation evokes severe impairments of cardiac performance

Next, we investigated the ECG and the blood pressure of freely moving mice by telemetric sensors to identify possible reasons underlying the lethal effect of CNO treatment on Dq⁺ mice (Figure 2). We found that only CNO injection into Dq⁺ mice caused cardiac arrhythmias (Figure 2A, bottom row, labelled with ‘Dq⁺ +CNO’), while neither CNO injection into WT, nor saline injection into WT or Dq⁺ mice had any measurable effect (Figure 2A, three upper rows). ECGs recorded before injections were superimposable between Dq⁺ and their WT littermates (Figure 2B, right panel). From a normal ECG before CNO injection (Figure 2B), ECG disorders manifested shortly after CNO administration of MCK-Dq mice as atrioventricular (AV) block Type I (Figure 2C), Type II (Figure 2D) with intermittent periods of ventricular tachycardia (with 15 Hz) and AV dissociation (Figure 2E) that eventually caused the death of the animal (data not shown). The statistical analysis of ECG key parameters clearly showed that the AV conduction time (PQ interval) increased over a 60-min time period only in the CNO-injected Dq⁺ mice (Figure 2F). In addition, impulse propagation within the ventricles was also distorted (prolonged QRS complex, Figure 2G), and repolarization of the ventricular tissue was substantially prolonged (QTc interval, Figure 2H) in these animals. Simultaneous monitoring of the mean arterial pressure (MAP) revealed that MAP increased from a basal value of 85.7±6.4 mmHg to 131.1±7.1 mmHg 5 min after CNO treatment of Dq⁺ mice (Figure 2I, red symbols and curve). Under all other conditions, mice displayed only a minor increase of around 10–15 mmHg and only a brief increase in their heart rate (Supplementary material online, Figure S5), most likely caused by the stress of the injection procedure. Interestingly, for the lower CNO dose in Dq⁺ mice, the changes in ECG parameters such as PR- and QRS duration were not different between animals displaying mortality and those surviving post-CNO application (Supplementary material online, Figure S6).

3.4 Ex vivo Dq activation induces severe alterations of the heart’s electrophysiology

In addition, we also carried out experiments on spontaneously beating hearts. Figure 3 summarizes key results of this analysis. In order to highlight changes in the surface ECGs recorded from Dq⁺ and WT hearts, we stacked individual ECG sweeps (synchronized to their R wave) in a way that the time within the sweep runs along the x-axis and the experimental time runs from the background into the foreground of the paper plane (Figure 3A and B). The CNO concentration in the perfusion solution was 7.5 µM, which corresponds to the blood plasma concentration found 15 min after CNO injection (10 mg/kg i.p.) in living mice (see above). The resulting surface plots of the ECG behaviour of a typical WT (Figure 3A) and Dq⁺ heart (Figure 3B) were colour-coded to highlight the observed changes. For specific experimental time points, individual ECG traces were replotted to the right of each surface plot. As observed in the in vivo experiments, the isolated Dq⁺ hearts also displayed substantial changes in their electrophysiological behaviour following CNO application. We analysed these changes for several hearts and found a rapid decrease of the R-wave amplitude (Figure 3C), a substantial increase of the PQ interval (Figure 3D), and an increased duration of the QRS (Figure 3E). These findings strongly suggest that the cardiac changes observed with CNO-treated Dq⁺ mice in vivo originated in the heart rather than being caused through indirect mechanisms involving other tissues.

Figure 3.

Ex vivo activation of Dq in whole hearts from Dq⁺ mice caused substantial electrophysiological changes. Hearts from Dq⁺ and WT mice were mounted on a Langendorff perfusion setup and ECG traces were recorded with surface electrodes. (A) Left: surface plot of all monophasic action potentials (synchronized to their R wave) from a WT heart during a 7-min experiment. A 7.5 µM CNO was applied after 1 min as indicated. The yellow dashed line connects the peaks of the P wave during the experiment. Right: representative single sweeps recorded at the time points given. Green lines mark the PQ interval. (B) Left: surface plot of ECG sweeps recorded from a Dq positive mouse heart. Prolongation of PQ interval and reduction in R-wave amplitude are visible. Right: representative single sweeps for the time points given. The green lines highlight the PQ interval. (C–E) Statistical analysis of the time course of the R-wave amplitude (C), PQ interval (D), and QRS duration (E) for WT (black) and Dq⁺ hearts (red). Each group comprised six hearts. Significant differences between Dq⁺ and WT hearts are indicated by a dashed line in the diagrams. Data in (C–E) were analysed with an unpaired t-test. Data points depict the mean values ± SEM.

3.5 Activation of Dq is positive inotropic in isolated myocytes

We next studied CNO-induced changes of electrically evoked Ca²⁺ transients in ventricular and atrial myocytes prepared from Dq⁺ mice. CNO-dependent activation of Dq in ventricular myocytes resulted in a positive inotropic response; the amplitude of electrically evoked Ca²⁺ transients increased by 40% (Figure 4A and B). Such inotropic responses were dependent on G_q-dependent signal transduction as indicated by their sensitivity to the G_q/11-specific inhibitor YM-254890^22–24 (Supplementary material online, Figure S7). Moreover, in model cells (HEK293) expressing the Dq receptor and either PIP2 or cAMP biosensors, CNO-dependent activation of Dq resulted in substantial PIP2 breakdown but not in accumulation of cAMP (Supplementary material online, Figure S8). Interestingly, the same CNO concentration (10 μM) applied to atrial myocytes evoked an increase of the amplitude of electrically evoked Ca²⁺ transients by 80% (Figure 4C). In addition, approximately 30% of all atrial myocytes were highly dysrhythmic 15 min after CNO treatment and could not be electrically paced anymore (see bottom traces Figure 4D). In the group of ventricular myocytes, only 2 cells (<5%) were dysrhythmic under similar conditions.

Figure 4.

Activation of Dq expressed by cardiac myocytes from Dq⁺ mice is positive inotropic and arrhythmogenic. Ca²⁺ responses of Fura-2 loaded, electrically paced mouse ventricular (A and B) and atrial (C and D) myocytes to the application of 10 µM CNO. Statistical analysis of the amplitudes of electrically evoked Ca²⁺ transients from Dq⁺ ventricular (A) or atrial (C) myocytes during vehicle (Ctrl, black) or CNO (10 µM, red) application at the time points given. Each bar represents 50–65 ventricular or 60–71 atrial myocytes from four animals. (A and C) After testing for normal distribution with a D’Agostino–Pearson omnibus normality test, the data were analysed with an unpaired t-test and the lines indicate the mean values ± SEM. Representative Ca²⁺ transients from Dq⁺ ventricular (B) or atrial (D) myocytes during control (black) recording or following the application of CNO (red) at the time points given. Please note that 30% of all atrial myocytes were dysrhythmic after 15 min after CNO administration and could not be paced anymore [lower row in (D)]. Arrowheads in (B) and (D) mark time points of field stimulation.

3.6 Activation of Dq does not alter the properties of cellular action potentials

Next, we examined whether Dq activation alters the shape of the cellular action potential and might thus directly contribute to the electrophysiological changes observed in vivo and in the isolated hearts (see above). Specifically, we employed the whole cell configuration of the patch clamp technique to address this question. Isolated ventricular myocytes from Dq⁺ mice were treated with 10 µm CNO for 6 min, and action potentials were studied before and after this time period (Figure 5). The ventricular action potential was not altered (Figure 5A and Ba–e) and the resting membrane potential remained unchanged (Figure 5Bf). These data suggested that the CNO-induced altered electrophysiological properties of the in vivo and ex vivo hearts were not caused by changes of the cellular action potential.

Figure 5.

Activation of Dq expressed by mouse ventricular myocytes from Dq⁺ mice does not alter cellular action potentials. Isolated ventricular myocytes were patch clamped in the whole cell configuration and subjected to current clamp measurements at 34°C. Action potentials were evoked by brief current injections at a rate of 2/s. (A) Displays typical action potentials of WT (left) and Dq⁺ ventricular myocytes under control conditions (black tracings) and 6 min after application of 10 µM CNO (red tracings). (B) Quantitative analysis of the various characteristic action potential durations (Ba–d), the action potential amplitude (Be), and the diastolic resting potential (Bf). For both conditions 22 ventricular myocytes from three animals of each genotype were analysed. After testing for normal distribution with a D’Agostino–Pearson omnibus normality test, the data were analysed with an unpaired t-test and the bars and errors indicate the mean values and SEM, respectively.

3.7 Dq co-localizes with Cx43 and its activation results in phosphorylation of CX43

We hypothesized that the heart’s electrophysiological changes might originate from altered cell–cell communication rather than solely from cellular alterations. To address this question, we investigated the spatial distribution of Dq and the major atrial and ventricular connexin isoform Cx43.²⁵ We found a strong co-localization of these two proteins (Figure 6A). This is visually emphasized in a 3D reconstruction of Dq and Cx43 immunofluorescence from confocal sections obtained from ventricular cryosections (Figure 6B). Areas of strong co-localization are highlighted by the yellow colour. For clarity, we highlighted a few intercalated discs (IDs) with white asterisks (Figure 6Ba). The part of the 3D volume from Figure 6Ba marked by the dashed box was re-plotted in Figure 6Bb at a higher magnification, and the same IDs were highlighted by the white asterisks.

Figure 6.

Dq co-localizes with Cx43 and its activation result in Cx43 phosphorylation. (A) Cryosections of ventricular tissue taken from Dq⁺ hearts were probed for Dq (Aa) and Cx43 (Ab). (Ac) Overlaid fluorescence to indicate co-localization is shown. The numbers in (Aa) depict the depth in the cryosections. (B) Three-dimensional reconstruction of the deconvolved, confocal z-stack (from A). Individual IDs can be identified and depict both, Dq and Cx43 immunoreactivity (indicated by the yellow colour). Individual IDs are marked by white asterisks in the yellow dashed box, which is re-plotted at a higher magnification in (Bb). Similar labelling was obtained from all cryosections (six per mouse heart, three mice). (C) CNO (10 mg/kg) was injected (i.p.) in vivo into Dq positive and WT mice. Hearts were excised, and total cardiac protein was extracted. While all mice depict positive Cx43 signals on western blots (Ca), phosphorylation at S368, a PKC-dependent process, only occurred in Dq positive mice (Cb). (Cc) Quantitative analysis of three experimental replicates depicting the normalized phosphorylation signal. Data were analysed with an unpaired t-test and the bars depict the mean values ± SEM.

The function and assembly of Cx43 is highly regulated by phosphorylation events^25–27 and PKC-mediated phosphorylation of Cx43 at position S368 has been reported to be critical for its physiological function²⁸ in cell–cell communication. We thus probed whether Dq activation leads to PKC-dependent S368 phosphorylation. We found that in vivo CNO application to Dq⁺ but not WT mice, resulted in a strong phosphorylation of Cx43 at position S368 (Figure 6Cb and d).

Finally, we probed the distribution and localization of Dq and Cx43 structures within individual IDs using instant structured illumination microscopy (iSIM) as depicted in Figure 7. We found that within individual IDs (asterisks in Figure 7Ab) Cx43 displays a patterned structure and that Dq shows a very good co-localization even within such ID substructures and might thus have privileged signalling access to cell–cell communication (Figure 7B).

Figure 7.

Dq co-localizes with Cx43 within individual IDs. iSIM-based super resolution analysis of Dq and Cx43 immunofluorescence in cryosections from Dq⁺ mice. (A) Two exemplified optical sections from a z-stack through a cryosection probed for Dq (green) and Cx43 (red) proteins (180 slices, 100 nm z-steps) that were 5 µm apart. The part of the z-stack marked by the dashed box in the left panel was volume rendered in (Ab). Individual IDs were marked by asterisks. The IDs labelled with the turquoise asterisks was replotted in (Ba–c) at a high magnification to illustrate the complex intra-IDs substructures. The left and right panel depict visualizations of the ID rotated around the z-axis. Similar results were found in all samples probed and analysed in this way (in total six cryosections from two Dq⁺ mice).

4. Discussion

Many cardiac processes are regulated by G-protein- and β-arrestin-dependent pathways. However, the relative contribution of these various pathways to cardiac physiology and pathophysiology in vivo has remained largely unexplored. Up to now, DREADD-based technologies are primarily used in the neuroscience field¹¹ with only few studies employing DREADDs in non-neuronal mammalian tissues.^15–17 DREADD technology has not been applied to study the function of striated muscle so far. Here, we describe a novel transgenic mouse model (MCK-Dq or Dq⁺ mouse) where a Gq-coupled DREADD (Dq) is expressed selectively in striated muscle.

The pharmacogenetic DREADD technology offers several advantages over optogenetic approaches. For example, in vivo activation of the pathway of interest can be performed non-invasively or with minimal manipulation (injection of the designer drug). In addition, DREADD-based studies do not require specialized equipment and mimic more closely physiologically occurring ligand-receptor interactions.

In the Dq⁺ mice, Dq expression was restricted to striated muscle including heart and skeletal muscle, in agreement with the specificity of the MCK promoter.²⁹ Consistent with the notion of a plasma membrane localization, Dq could not be detected in the cytosol but was enriched in the membrane fraction (Figure 1C), a finding that was substantiated in immunohistochemical studies (Figure 1D, Supplementary material online, Figure S1). These findings indicated that Dq expression and localization were similar to those of endogenous GPCRs.

In the absence of the DREADD agonist CNO, Dq⁺ mice showed no obvious cardiac phenotypes (Supplementary material online, Figure S3), indicating that Dq appears devoid of constitutive activity, as described earlier.¹⁵ In a study of in vivo consequences of Dq activation CNO treatment of Dq⁺ but not of WT mice, led to a dose-dependent mortality (100% at 10 mg/kg CNO) within a few hours (Supplementary material online, Figure S4). Dq activation caused phosphoinositide breakdown without measurable generation of cAMP¹⁵ (see also Supplementary material online, Figure S8).

Mimicking physiological Gq activation, CNO-dependent activation of Dq resulted in mortal cardiac arrhythmias together with a transient positive inotropy only in Dq⁺ mice (Figure 2). Such fatal events were always preceded by severe alterations in the shape and time course of the ECG, indicating that Gq activation did not only alter cardiac inotropy but also changed impulse propagation in the AV node (AV blocks of various degrees, Figure 2C and D) and spatiotemporal dispersion of the excitation wave in the ventricle (aberrant and prolonged QRS complex, Figure 2G). Interestingly, such alterations of the in vivo ECG were not different when measured in surviving or ceasing animals at the lower CNO concentration (Supplementary material online, Figure S6), suggesting that already the lower stimulation strength caused severe disturbances of impulse propagation. In addition, initiation of ventricular tachycardia and fibrillation however requires focal firing³⁰ most likely occurring with a lesser probability at the lower CNO concentration.

We confirmed that the cardiac malfunction observed with CNO-treated Dq⁺ mice did indeed originate in the heart and demonstrated that all cardiac malfunction that we observed in vivo was also found with CNO-treated hearts isolated from Dq⁺ mice (Figure 3).

Activation of Gq signalling in isolated cardiomyocytes was reported to cause increases in the amplitude of electrically evoked Ca²⁺ transients in ventricular³¹ and atrial myocytes.^32,33 The latter was accompanied by a pronounced occurrence of spontaneous Ca²⁺ sparks at locations of ryanodine receptor/inositol-1,4,5-trisphophoate receptor (InsP₃R) co-localization causing the occurrence of delayed after depolarizations (DADs).³⁴ Consistent with this observation, CNO treatment of Dq⁺ ventricular and atrial myocytes evoked a substantial inotropic response (Figure 4). Dq-mediated activation of Gq signalling only caused substantial dysrhythmia in atrial myocytes. Such effects are likely to involve the activation of InsP₃R in the heart.^32–34

The occurrence of cellular inotropy appeared strictly G_q-dependent (Supplementary material online, Figure S7). Activation of G_q signalling results in the liberation of two second messengers, DAG and InsP₃ and both branches are likely to contribute to the inotropic responses. While InsP₃ might activate neighbouring ryanodine receptors, thereby increase the amplitude of cellular Ca²⁺ transients, DAG can stimulate a plethora of downstream signalling events, the most prominent being the activation of a member of the PKC family. PKCs are known to phosphorylate proteins involved in inotropic responses, including the ryanodine receptor,³⁵ L-type Ca²⁺ channels,³⁶ and the cardiac myosin-binding protein C,³⁷ suggesting a multitude of possible mechanisms for the cellular inotropy and the increase of the MAP measured in vivo (see Figure 2). In addition, recent studies have highlighted the possibility for additional Ca²⁺ influx pathways that contribute to increased SR-Ca²⁺load during neurohumoral stimulation.³⁸

Our data from in vivo ECG and whole heart surface potential recordings indicated that Dq-mediated activation of Gq signalling resulted in pronounced and immediate changes of the electrical properties of the heart, causing substantial alterations of impulse propagation (Figures 2 and 3), without changes of cellular action potential (Figure 5). Dq strongly co-localizes with the major cardiac connexin, Cx43 (Figure 6) at the level of individual IDs. Super-resolution imaging revealed that these two proteins even co-localized on substructures of IDs probably resembling the electron-dense plaques described recently.³⁹ Importantly, activation of Dq in vivo resulted in substantial Cx43 phosphorylation at S368, a site whose phosphorylation was described to be PKC-dependent,^25,28 a kinase family that is activated downstream of Gq-signalling. S368 phosphorylation has been implicated in aberrant connexon assembly and function, such as ion conductivity.^26–28 Thus, our findings support the novel concept that Gq stimulation not only alters the cell’s calcium handling but, most likely, has a direct and immediate effect on the properties of connexins, resulting in aberrant impulse generation, propagation and dispersion in the heart.

5. Conclusion

In conclusion, the occurrence of Gq-dependent arrhythmias, as described in the current report, might result from the two branches that appear to be necessary for their initiation and manifestation; (i) generation of a substrate (inhomogeneous excitability) and (ii) focal firing as reviewed for atrial fibrillation³⁰ and ventricular arrhythmias.⁴⁰ It appears feasible to assume that Gq signalling is an important contributor to both branches. InsP₃ signalling has been shown to be a potent initiator for the generation of DADs^32,33 that might serve the role of ‘focal firing’.³⁰ This spontaneous firing alone will generate inhomogeneous excitability in the tissue. The reported Gq-dependent, PKC-mediated phosphorylation of the major cardiac connexin isoform, Cx43, has been associated with malfunction of intercellular communication resulting in slow impulse propagation,⁴¹ a putative prerequisite for inhomogeneous excitability and the occurrence of re-entry phenomena.³⁰ One might speculate that phosphorylation of the other major cardiac connexins Cx40 and Cx45 also occurs following Gq-activation and additionally contributes to the aforementioned mechanisms.⁴¹

There is room for improvements of the current mouse model. We cannot completely exclude the possibility that CNO activation of Dq receptors expressed by skeletal muscle indirectly modulates cardiac function. A possible way to circumvent this problem would be the use of Cre-recombinase dependent Dq expression systems, as described recently.⁹

In summary, we demonstrate, that the pharmacogenetic DREADD technology is highly useful to explore the outcome of enhanced Gq signalling in the heart and made the novel observation that acute Gq-signalling in the heart immediately triggers aberrant impulse penetration, propagation, and dispersion. The latter effects might be a result of Dq-Gq-PKC mediated connexin 43 phosphorylation. Use of the newly generated Dq⁺ mice will enable researchers, for the first time, to further study the consequences of selective activation of cardiac Gq signalling in vivo.

Time for primary review: 43 days