LRP6 modulates the phosphorylation of Cx43 via Gαs in ventricular tachycardia of myocardial infarction

Background Ventricular tachycardia (VT) and ventricular fibrillation are the most causes of early death in patients with myocardial infarction (MI). This study was aimed to explore whether LRP6 and its upstream genes circRNA1615 and miR-152-3p modulated the phosphorylation of Connexin-43 (Cx43) via Gαs in ventricular tachycardia of MI. Method we constructed the hypoxia cardiomyocyte model and AMI mice, and explored the modulation relationship of LRP6 and its upstream genes circRNA1615 and miR-152-3p. In addition, the immunoblot analysis with monoclonal and polyclonal antibodies were used to detect whether LRP6 and Cx43 were phosphorylated, further investigated that the LRP6 regulated the phosphorylation of its downstream target Cx43 via G-protein alpha subunit Gαs by using cell transfection, FISH assay, HE staining, RT-qPCR, and Western blot techniques. Result LRP6 mRNA expression was significantly reduced in AMI group compared with the control group. Hypoxia could inhibit the protein and phosphorylation levels of LRP6 and Cx43. The expression of circRNA1615 in AMI mice was significantly decreased, but overexpression of circRNA1615 significantly reversed it. Also overexpression of circRNA1615 could weaken the effect of miR-152-3p mimic, and the miR-152-3p mimic increased the hypoxia injury of LRP6 and Cx43, further LRP6 interference fragments could aggravate hypoxia injury of Cx43. The overexpression of LRP6 could significantly increase the protein level and phosphorylation level of Cx43, but the interference with LRP6 showed the opposite trend. Noticeably, the interference with Gαs weakened the protein and phosphorylation levels of Cx43, however, the interference with LRP6 further inhibited the protein and phosphorylation levels of Cx43. Finally, the transcriptions of circRNA1615 and LRP6 were inhibited in AMI, but the transcription of miR-152-3p was promoted, and the overexpression of circRNA1615 could weaken the damage effect and VT of AMI. Conclusion LRP6 and its upstream genes circRNA1615 and miR-152-3p modulated the phosphorylation of Cx43 via Gαs in ventricular tachycardia of myocardial infarction.

Gsα deficiency facilitates cardiac remodeling via CREB/ Bmp10-mediated signaling

The stimulatory G-protein alpha subunit (Gsα), a ubiquitously expressed protein, mediates G-protein receptor-stimulated signal transduction. To investigate the functions of Gsα in cardiomyocytes. We developed transverse aortic constriction (TAC)-induced heart failure mouse models and tamoxifen-inducible transgenic mice with cardiac-specific Gsα disruption. We detected alterations in Gsα expression in TAC-induced heart failure mice. Moreover, we examined cardiac function and structure in mice with genetic Gsα deletion and investigated the underlying molecular mechanisms of Gsα function. We found that Gsα expression increased during the compensated cardiac hypertrophy period and decreased during the heart failure period. Moreover, cardiac-specific Gsα disruption deteriorated cardiac function and induced severe cardiac remodeling. Mechanistically, Gsα disruption decreased CREB1 expression and inhibited the Bmp10-mediated signaling pathway. In addition, we found that Gsα regulates Bmp10 expression through the binding of CREB1 to the Bmp10 promoter. Our results suggest that fluctuations in Gsα levels may play a vital role in the development of heart failure and that loss of Gsα function facilitates cardiac remodeling.

Lack of GNAS re-methylation during oogenesis may be a cause of sporadic pseudohypoparathyroidism type Ib (PHP1B)

Context Pseudohypoparathyroidism type Ib (PHP1B) is characterized by hypocalcemia and hyperphosphatemia due to PTH-resistance in the proximal renal tubules. Maternal pathogenic STX16/GNAS variants leading to maternal epigenetic GNAS changes impair expression of the stimulatory G protein alpha-subunit (Gsα) thereby causing autosomal dominant PHP1B (AD-PHP1B). In contrast, genetic defects responsible for sporadic PHP1B (sporPHP1B) remain mostly unknown. Objective Determine whether PHP1B encountered after in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI) causes GNAS re-methylation defects similar to those in sporPHP1B. Design Retrospective analysis. Results Nine among thirty-six sporPHP1B patients investigated since 2000, all with LOM at the three maternal GNAS DMRs and gain-of-methylation (GOM) at the paternal NESP DMR, had been conceived through IVF or ICSI. Besides abnormal GNAS methylation, IVF/ICSI-PHP1B cases revealed no additional imprinting defects. Three of these PHP1B patients have dizygotic twins and four have IVF/ICSI-conceived siblings, all with normal GNAS methylation; two unaffected younger siblings were conceived naturally. Conclusion Sporadic and IVF/ICSI-conceived PHP1B patients revealed indistinguishable epigenetic changes at all four GNAS DMRs thus suggesting a similar underlying disease mechanism. Given that re-methylation at the three maternal DMRs occurs during oogenesis, male factors are unlikely to cause LOM post-fertilization. Instead, at least some of the sporPHP1B variants could be caused by a defect(s) in an oocyte-expressed gene that is required for fertility and for re-establishing maternal GNAS methylation imprints. It remains uncertain, however, whether lack of GNAS re-methylation alone impairs oocyte maturation because of insufficient Gsα expression, thus necessitating Assisted Reproductive Technology (ART) for conception.

Mice With Monoallelic GNAO1 Loss Exhibit Reduced Inhibitory Synaptic Input To Cerebellar Purkinje Cells

GNAO1 encodes Gαo, a heterotrimeric G protein alpha subunit in the Gi/o family. In this report, we used a Gnao1 mouse model G203R previously described as a gain-of-function Gnao1 mutant with movement abnormalities and enhanced seizure susceptibility. Here, we report an unexpected second mutation resulting in a loss-of-function Gαo protein and describe alterations in central synaptic transmission. Whole cell patch clamp recordings from Purkinje cells (PCs) in acute cerebellar slices from Gnao1 mutant mice showed significantly lower frequencies of spontaneous and miniature inhibitory postsynaptic currents (sIPSCs and mIPSCs) compared to WT mice. There was no significant change in sEPSCs or mEPSCs. Whereas mIPSC frequency was reduced, mIPSC amplitudes were not affected, suggesting a presynaptic mechanism of action. A modest decrease in the number of molecular layer interneurons was insufficient to explain the magnitude of IPSC suppression. Paradoxically, Gi/o inhibitors (pertussis toxin), enhanced the mutant-suppressed mIPSC frequency and eliminated the difference between WT and Gnao1 mice. While GABAB receptor regulates mIPSCs, neither agonists nor antagonists of this receptor altered function in the mutant mouse PCs. This study is the first electrophysiological investigation of the role of Gi/o protein in cerebellar synaptic transmission using an animal model with a loss-of-function Gi/o protein.

Maternal GNAS Contributes to the Extra-Large G Protein α-Subunit (XLαs) Expression in a Cell Type-Specific Manner

GNAS encodes the stimulatory G protein alpha-subunit (Gsα) and its large variant XLαs. Studies have suggested that XLαs is expressed exclusively paternally. Thus, XLαs deficiency is considered to be responsible for certain findings in patients with paternal GNAS mutations, such as pseudo-pseudohypoparathyroidism, and the phenotypes associated with maternal uniparental disomy of chromosome 20, which comprises GNAS. However, a study of bone marrow stromal cells (BMSC) suggested that XLαs could be biallelically expressed. Aberrant BMSC differentiation due to constitutively activating GNAS mutations affecting both Gsα and XLαs is the underlying pathology in fibrous dysplasia of bone. To investigate allelic XLαs expression, we employed next-generation sequencing and a polymorphism common to XLαs and Gsα, as well as A/B, another paternally expressed GNAS transcript. In mouse BMSCs, Gsα transcripts were 48.4 ± 0.3% paternal, while A/B was 99.8 ± 0.2% paternal. In contrast, XLαs expression varied among different samples, paternal contribution ranging from 43.0 to 99.9%. Sample-to-sample variation in paternal XLαs expression was also detected in bone (83.7–99.6%) and cerebellum (83.8 to 100%) but not in cultured calvarial osteoblasts (99.1 ± 0.1%). Osteoblastic differentiation of BMSCs shifted the paternal XLαs expression from 83.9 ± 1.5% at baseline to 97.2 ± 1.1%. In two human BMSC samples grown under osteoinductive conditions, XLαs expression was also predominantly monoallelic (91.3 or 99.6%). Thus, the maternal GNAS contributes significantly to XLαs expression in BMSCs but not osteoblasts. Altered XLαs activity may thus occur in certain cell types irrespective of the parental origin of a GNAS defect.

The protection of Gαz-null NOD mice from hyperglycemia is sexually dimorphic and only partially β-cell autonomous

The mechanisms that underlie the β-cell pathophysiology of Type 1 Diabetes (T1D) are not fully understood. Our group has defined the unique heterotrimeric G protein alpha-subunit, Gαz, as a key negative regulator of β-cell signal transduction pathways. Non-obese diabetic (NOD) mice lacking Gαz throughout the body are protected from developing T1D-like hyperglycemia. To determine whether this phenotype is β-cell autonomous, we generated and validated a β-cell-specific Gαz knockout (βKO) on the NOD background and characterized the phenotype of female and male cohorts. Long-term hyperglycemia incidence was lower in Gαz βKO mice as compared to wild-type (WT) controls, but, unlike global Gαz knockout mice, this protection was incomplete. While young male and female Gαz βKO NOD mice had improved glucose tolerance, WT NOD males were significantly less glucose tolerant than females, and only female Gαz βKO mice retained improved glucose tolerance at 28-29 weeks of age. Conversely, β-cell-specific Gαz loss only influenced insulitis in 28-29-week old male NOD mice, a phenotype correlating directly with body burden of glucose during oral glucose challenge. Using surrogates for β-cell function and apoptosis, the partial penetrance of euglycemia in Gαz βKO NOD was best explained by an early failure to up-regulate β-cell proliferation. We conclude β-cell Gαz is an important regulator of the sexually-dimorphic T1D-like phenotype of NOD mice. Yet, other factors must be important in imparting full protection from the disease.

Heterotrimeric G-protein α subunit (RGA1) regulates tiller development, yield, cell wall, nitrogen response and biotic stress in rice

G-proteins are implicated in plant productivity, but their genome-wide roles in regulating agronomically important traits remain uncharacterized. Transcriptomic analyses of rice G-protein alpha subunit mutant (rga1) revealed 2270 differentially expressed genes (DEGs) including those involved in C/N and lipid metabolism, cell wall, hormones and stress. Many DEGs were associated with root, leaf, culm, inflorescence, panicle, grain yield and heading date. The mutant performed better in total weight of filled grains, ratio of filled to unfilled grains and tillers per plant. Protein–protein interaction (PPI) network analysis using experimentally validated interactors revealed many RGA1-responsive genes involved in tiller development. qPCR validated the differential expression of genes involved in strigolactone-mediated tiller formation and grain development. Further, the mutant growth and biomass were unaffected by submergence indicating its role in submergence response. Transcription factor network analysis revealed the importance of RGA1 in nitrogen signaling with DEGs such as Nin-like, WRKY, NAC, bHLH families, nitrite reductase, glutamine synthetase, OsCIPK23 and urea transporter. Sub-clustering of DEGs-associated PPI network revealed that RGA1 regulates metabolism, stress and gene regulation among others. Predicted rice G-protein networks mapped DEGs and revealed potential effectors. Thus, this study expands the roles of RGA1 to agronomically important traits and reveals their underlying processes.

Correction to: Interaction of the heterotrimeric G protein alpha subunit SSG-1 of Sporothrix schenckii with proteins related to stress response and fungal pathogenicity using a yeast two-hybrid assay

Following the publication of this article [1], it was brought to our attention that Fig. 7A lane 2 is identical to Fig. 7B lane 2 and Fig. 7B lane 4 is identical to Fig. 7C lane 4.

Extra-Large Gα Protein (XLαs) Deficiency Causes Severe Adenine-Induced Renal Injury with Massive FGF23 Elevation

Fibroblast growth factor-23 (FGF23) is critical for phosphate and vitamin D homeostasis. Cellular and molecular mechanisms underlying FGF23 production remain poorly defined. The extra-large Gα subunit (XLαs) is a variant of the stimulatory G protein alpha-subunit (Gsα), which mediates the stimulatory action of parathyroid hormone in skeletal FGF23 production. XLαs ablation causes diminished FGF23 levels in early postnatal mice. Herein we found that plasma FGF23 levels were comparable in adult XLαs knockout (XLKO) and wild-type littermates. Upon adenine-rich diet-induced renal injury, a model of chronic kidney disease, both mice showed increased levels of plasma FGF23. Unexpectedly, XLKO mice had markedly higher FGF23 levels than WT mice, with higher blood urea nitrogen and more severe tubulopathy. FGF23 mRNA levels increased substantially in bone and bone marrow in both genotypes; however, the levels in bone were markedly higher than in bone marrow. In XLKO mice, a positive linear correlation was observed between plasma FGF23 and bone, but not bone marrow, FGF23 mRNA levels, suggesting that bone, rather than bone marrow, is an important contributor to severely elevated FGF23 levels in this model. Upon folic acid injection, a model of acute kidney injury, XLKO and WT mice exhibited similar degrees of tubulopathy; however, plasma phosphate and FGF23 elevations were modestly blunted in XLKO males, but not in females, compared to WT counterparts. Our findings suggest that XLαs ablation does not substantially alter FGF23 production in adult mice but increases susceptibility to adenine-induced kidney injury, causing severe FGF23 elevations in plasma and bone.