Inhibition of G-protein signalling in cardiac dysfunction of intellectual developmental disorder with cardiac arrhythmia (IDDCA) syndrome

Abstract

Background: Pathogenic variants of GNB5 encoding the β₅ subunit of the guanine nucleotide-binding protein cause IDDCA syndrome, an autosomal recessive neurodevelopmental disorder associated with cognitive disability and cardiac arrhythmia, particularly severe bradycardia.

Methods: We used echocardiography and telemetric ECG recordings to investigate consequences of Gnb5 loss in mouse.

Results: We delineated a key role of Gnb5 in heart sinus conduction and showed that Gnb5-inhibitory signalling is essential for parasympathetic control of heart rate (HR) and maintenance of the sympathovagal balance. Gnb5^-/- mice were smaller and had a smaller heart than Gnb5^+/+ and Gnb5^+/- , but exhibited better cardiac function. Lower autonomic nervous system modulation through diminished parasympathetic control and greater sympathetic regulation resulted in a higher baseline HR in Gnb5^-/- mice. In contrast, Gnb5^-/- mice exhibited profound bradycardia on treatment with carbachol, while sympathetic modulation of the cardiac stimulation was not altered. Concordantly, transcriptome study pinpointed altered expression of genes involved in cardiac muscle contractility in atria and ventricles of knocked-out mice. Homozygous Gnb5 loss resulted in significantly higher frequencies of sinus arrhythmias. Moreover, we described 13 affected individuals, increasing the IDDCA cohort to 44 patients.

Conclusions: Our data demonstrate that loss of negative regulation of the inhibitory G-protein signalling causes HR perturbations in Gnb5^-/- mice, an effect mainly driven by impaired parasympathetic activity. We anticipate that unravelling the mechanism of Gnb5 signalling in the autonomic control of the heart will pave the way for future drug screening.

Keywords: GNB5variants; Gnb5-null mouse models; IDDCA; cardiac conduction anomalies.

Figure 1

Variants modelling and IDDCA mutational spectrum. (A, top left) Top view of the Gnb5 (orange, PDB entry 2pbi) 3D protein model, showing the mutated Trp81 (yellow) and the glycerol molecule (green) in the centre of the pore. The rearrangements necessary to accommodate a tryptophan residue at position 81 will change the channel characteristics. The rotamer displayed here highlights clashes of Trp81 with Cys68 (white) and Cys111 (magenta). In additional rotamers, the bulky tryptophan sidechain will severely bump into Leu67 (grey), Val87 (pink), Val108 (cyan) and Ala110 (brown). (A, top right) As shown in this view of the beta propeller from above, the ‘wild-type’ Gly215 (not shown) lays in a beta-sheet, with on top Ala221 (pink), at the bottom Cys200 (magenta), and in front a beta-strand harbouring Val242 (grey) and Val245 (cyan). The presence of Glu215 cannot be tolerated, as it will encroach into one of the residues previously enumerated. Another rotamer shows clashes into Val245 (cyan). Overall, all rotamers may also force the sidechain of Cys200 (magenta) to reorient itself toward the internal part of the channel to provide space to accommodate glutamine at position 215 (yellow). In this position, the Cys200 sidechain will occupy the space dedicated to the glycerol (green), thus changing the properties of the channel. (A, bottom) The Leu59 (purple label, left panel) is positioned closely to the Leu349 residue just above (red label) in an antiparallel beta-sheet. This secondary structure will likely be broken in presence of a proline at that position (blue label, right panel), which in turn will disrupt the protein folding as the proline sidechain will collide into Leu349 (red label). (B) Distribution of the IDDCA published and novel variants along the schematically represented 11 exons of the human GNB5 gene (transcript NM_006578.3; Ensembl (release 98, September 2019). The variants of IDDCA affected individuals are represented in red (LoF) and the missense LADCI variants in blue. The yellow star marks the variant of Middle Eastern descent, while green stars indicate the amber and ochre variants from the Indian subcontinent (dark green) and of Pakistani descent (light green), respectively. IDDCA, intellectual developmental disorder with cardiac arrhythmia; LADCI, language delay and ADHD/cognitive impairment; LoF, loss of function.

Figure 2

Gnb5 mouse line features. (A) Mouse mating strategy and gender and genotype distribution over three successive generations. Preweaning and postweaning mortality is reported for each colony. (B) Size of Gnb5^+/+ and Gnb5^{−/ −} mice. (C, D) Body weights profile monitored from 3–46 weeks of age. All mice were weaned on week 3. Data are shown as mean±SD. (C, D) Panels separate body weights according to sex. Gnb5^+/+ is depicted in grey, Gnb5^{+/ −} in blue and Gnb5^{−/ −} in red. (E) At sacrifice, neither significant morphology difference nor thoracic position of the heart were observed among groups. (F) Bar plot showing that Gnb5^{−/ −} hearts (red, n=16) are smaller compared with the other genotypes (Gnb5^{+/ −}, blue (n=8) and Gnb5^+/+, grey (n=16)). Data are shown as mean of the ratio between heart weight and tibia length (used to normalise for animal size)±SEM. Asterisks on the plots represent the level of significance: *p≤0.05, **p≤0.01, ***p≤0.001, ****p≤0.0001; p>0.05 was considered not significant.

Figure 3

Morphological and functional parameters measured by ultrasound scan in Gnb5^+/+ , Gnb5^{+/ −} and Gnb5^{−/ −} male mice. (A) Left ventricular internal diameter at diastole (left) and systole (right). (B) Left ventricular volume in diastole (left) and systole (right). (C) Mass of the left ventricle. (D–G) Cardiac function expressed as fractional shortening (D), ejection fraction (E), cardiac output (F) and stroke volume (G). Parameters unchanged between the three genotypes are shown in online supplemental figure S2A, B.

Figure 4

Cardiac arrhythmias recorded in Gnb5 mouse line. (A) Normal ECG trace recorded in wild-type male mice with the main spikes specified as used in the text. (B, C) Gnb5^−/− male mice ECG traces showing escape atrial beats classified as short (B) and long (C) and characterised by the occurrence of a late P-wave (red arrow). (D) Gnb5^{−/ −} male mice ECG trace demonstrating atrioventricular blocks, with more than one P-wave per QRS complex (consecutive red arrows). (E–G) Respective box plots indicating the number of arrhythmias, that is, the number of short (E) and long (F) escape atrial beats and atrioventricular blocks (G) per 24 hours. ns, not significant.

Figure 5

Pharmacological administration of compounds mimicking parasympathetic and sympathetic stimulation. (A) HR monitoring after injection of atropin (intraperitoneal 1 mg/kg). (B) Bradycardia measured in response to carbachol (intraperitoneal 0.1 m/kg). (C) HR variation in response to atenolol (intraperitoneal 2 mg/kg). (D) Increased HR after isoprenaline administration (intraperitoneal 4 mg/kg). Data points are expressed as percentage of the baseline values. (E) Smaller parasympathetic and bigger sympathetic blockade in Gnb5^−/− mice (red), compared with wild-type littermates (black), indicative of lower parasympathetic and higher sympathetic tones in basal conditions. HR, heart rate.

Figure 6

Comparison of transcriptome profiles of atria and ventricles in Gnb5^+/+ , Gnb5^+/ and Gnb5^−/− male mice. (A) Atrial expression profiles of Gnb5 (top, left) and other DEGs (top Lane); Gnb and Rgs transcripts quantification in atria (bottom lane). (B) Ventricular expression profiles of Gnb5 gene (top, left) and other DEGs (top lane); Gnb and Rgs transcripts quantification in ventricles (bottom). DEG, differentially expressed gene.