Conditional disruption of interactions between Gαi2 and regulator of G protein signaling (RGS) proteins protects the heart from ischemic injury

Abstract Background Regulator of G protein signaling (RGS) proteins suppress G protein coupled receptor signaling by catalyzing the hydrolysis of Gα-bound guanine nucleotide triphosphate. Transgenic mice in which RGS-mediated regulation of Gαi2 is lost (RGS insensitive Gαi2 G184S) exhibit beneficial (protection against ischemic injury) and detrimental (enhanced fibrosis) cardiac phenotypes. This mouse model has revealed the physiological significance of RGS/Gαi2 interactions. Previous studies of the Gαi2 G184S mutation used mice that express this mutant protein throughout their lives. Thus, it is unclear whether these phenotypes result from chronic or acute Gαi2 G184S expression. We addressed this issue by developing mice that conditionally express Gαi2 G184S. Methods Mice that conditionally express RGS insensitive Gαi2 G184S were generated using a floxed minigene strategy. Conditional expression of Gαi2 G184S was characterized by reverse transcription polymerase chain reaction and by enhancement of agonist-induced inhibition of cAMP production in isolated cardiac fibroblasts. The impact of conditional RGS insensitive Gαi2 G184S expression on ischemic injury was assessed by measuring contractile recovery and infarct sizes in isolated hearts subjected to 30 min ischemia and 2 hours reperfusion. Results We demonstrate tamoxifen-dependent expression of Gαi2 G184S, enhanced inhibition of cAMP production, and cardioprotection from ischemic injury in hearts conditionally expressing Gαi2 G184S. Thus the cardioprotective phenotype previously reported in mice expressing Gαi2 G184S does not require embryonic or chronic Gαi2 G184S expression. Rather, cardioprotection occurs following acute (days rather than months) expression of Gαi2 G184S. Conclusions These data suggest that RGS proteins might provide new therapeutic targets to protect the heart from ischemic injury. We anticipate that this model will be valuable for understanding the time course (chronic versus acute) and mechanisms of other phenotypic changes that occur following disruption of interactions between Gαi2 and RGS proteins.http://deepblue.lib.umich.edu/bitstream/2027.42/109553/1/40360_2014_Article_315.pd

Pleiotropic Phenotype of a Genomic Knock-In of an RGS-Insensitive G184S Gnai2 Allele

Signal transduction via guanine nucleotide binding proteins (G proteins) is involved in cardiovascular, neural, endocrine, and immune cell function. Regulators of G protein signaling (RGS proteins) speed the turn-off of G protein signals and inhibit signal transduction, but the in vivo roles of RGS proteins remain poorly defined. To overcome the redundancy of RGS functions and reveal the total contribution of RGS regulation at the Gα(i2) subunit, we prepared a genomic knock-in of the RGS-insensitive G184S Gnai2 allele. The Gα(i2)(G184S) knock-in mice show a dramatic and complex phenotype affecting multiple organ systems (heart, myeloid, skeletal, and central nervous system). Both homozygotes and heterozygotes demonstrate reduced viability and decreased body weight. Other phenotypes include shortened long bones, a markedly enlarged spleen, elevated neutrophil counts, an enlarged heart, and behavioral hyperactivity. Heterozygous Gα(i2)(+/G184S) mice show some but not all of these abnormalities. Thus, loss of RGS actions at Gα(i2) produces a dramatic and pleiotropic phenotype which is more evident than the phenotype seen for individual RGS protein knockouts

Regulator of G Protein Signaling Protein Suppression of Gαo Protein-Mediated α2A Adrenergic Receptor Inhibition of Mouse Hippocampal CA3 Epileptiform Activity

Activation of G protein-coupled α2 adrenergic receptors (ARs) inhibits epileptiform activity in the hippocampal CA3 region. The specific mechanism underlying this action is unclear. This study investigated which subtype(s) of α2ARs and G proteins (Gαo or Gαi) are involved in this response using recordings of mouse hippocampal CA3 epileptiform bursts. Application of epinephrine (EPI) or norepinephrine (NE) reduced the frequency of bursts in a concentration-dependent manner: (-)EPI > (-)NE >>> (+)NE. To identify the α2AR subtype involved, equilibrium dissociation constants (pKb) were determined for the selective αAR antagonists atipamezole (8.79), rauwolscine (7.75), 2-(2,6-dimethoxyphenoxyethyl)aminomethyl-1,4-benzodioxane hydrochloride (WB-4101; 6.87), and prazosin (5.71). Calculated pKb values correlated best with affinities determined previously for the mouse α2AAR subtype (r = 0.98, slope = 1.07). Furthermore, the inhibitory effects of EPI were lost in hippocampal slices from α2AAR-but not α2CAR-knockout mice. Pretreatment with pertussis toxin also reduced the EPI-mediated inhibition of epileptiform bursts. Finally, using knock-in mice with point mutations that disrupt regulator of G protein signaling (RGS) binding to Gα subunits to enhance signaling by that G protein, the EPI-mediated inhibition of bursts was significantly more potent in slices from RGS-insensitive GαoG184S heterozygous (Gαo+/GS) mice compared with either Gαi2G184S heterozygous (Gαi2+/GS) or control mice (EC50 = 2.5 versus 19 and 23 nM, respectively). Together, these findings indicate that the inhibitory effect of EPI on hippocampal CA3 epileptiform activity uses an α2AAR/Gαo protein-mediated pathway under strong inhibitory control by RGS proteins. This suggests a possible role for RGS inhibitors or selective α2AAR agonists as a novel antiepileptic drug therapy