Protection of cardiomyocytes from the hypoxia-mediated injury by a peptide targeting the activator of G-protein signaling 8.

Signaling via heterotrimeric G-protein is involved in the development of human diseases including ischemia-reperfusion injury of the heart. We previously identified an ischemia-inducible G-protein activator, activator of G-protein signaling 8 (AGS8), which regulates Gβγ signaling and plays a key role in the hypoxia-induced apoptosis of cardiomyocytes. Here, we attempted to intervene in the AGS8-Gβγ signaling process and protect cardiomyocytes from hypoxia-induced apoptosis with a peptide that disrupted the AGS8-Gβγ interaction. Synthesized AGS8-peptides, with amino acid sequences based on those of the Gβγ-binding domain of AGS8, successfully inhibited the association of AGS8 with Gβγ. The AGS8-peptide effectively blocked hypoxia-induced apoptosis of cardiomyocytes, as determined by DNA end-labeling and an increase in cleaved caspase-3. AGS8-peptide also inhibited the change in localization/permeability of channel protein connexin 43, which was mediated by AGS8-Gβγ under hypoxia. Small compounds that inhibit a wide range of Gβγ signals caused deleterious effects in cardiomyocytes. In contrast, AGS8-peptide did not cause cell damage under normoxia, suggesting an advantage inherent in targeted disruption of the AGS8-Gβγ signaling pathway. These data indicate a pivotal role for the interaction of AGS8 with Gβγ in hypoxia-induced apoptosis of cardiomyocytes, and suggest that targeted disruption of the AGS8-Gβγ signal provides a novel approach for protecting the myocardium against ischemic injury

Determination of Gβγ interacting domain of the AGS8.

<p>(A) Schematic diagram of rat AGS8 and the AGS8 domains synthesized as GST-fusion proteins. Each GST protein fused with the following segment of rat AGS8 (ABB82299) respectively. AGS8-N: M¹- P³⁷⁰, AGS8-254: A²⁵⁴– R⁵⁵³, AGS8-534: S⁵³⁴– S⁸³³, AGS8-814: S⁸¹⁴– R¹¹¹³, AGS8-1094: H¹⁰⁹⁴– D³²⁸⁰, AGS8-C: A¹³⁵⁹– W¹⁷³⁰. (B) GST-pulldown assay of AGS8 domains with Gβ₁γ₂. AGS8 domains synthesized as GST-fusion proteins (300 nM) were incubated with recombinant human Gβ₁γ₂ (30 nM) in a total volume of 300 µl at 4°C. Proteins were then adsorbed to a glutathione matrix and retained G-protein subunits identified by immunoblotting following gel electrophoresis. The representative of 5 independent experiments with similar results. (C) Bioactivity of AGS8 domains on G-protein activation in cell. The yeast strain expressing human Gαs was transformed with AGS8 domains described in (A) into the pYES2-containing GAL1 promoter. The yeast strain was modified to grow without histidine on activation of G-protein. Induction(+): induction of translation of AGS8 domains by galactose. The representative of 4 independent experiments with similar results. (D) GST-pull down assay of AGS8-C segments with recombinant Gβγ. (<i>upper panel</i>) Schematic diagram of AGS8-C and the segments synthesized as GST-fusion proteins. Each GST protein fused with the following segment of rat AGS8 (ABB82299) respectively. AGS8-C1: A¹³⁵⁹– H¹⁴⁹³, AGS8-C2: A¹⁴⁹⁴– T¹⁵⁸⁵. (<i>lower panel</i>) GST-pulldown assay of AGS8 segments with Gβ₁γ₂. AGS8 domains synthesized as GST-fusion proteins (300 nM) were incubated with recombinant human Gβ₁γ₂ (30 nM) in a total volume of 300 µl at 4°C. Proteins were then adsorbed to a glutathione matrix and retained G-protein subunits identified by immunoblotting following gel electrophoresis. The representative of 4 independent experiments with similar results.</p

Effect of deletion of 45 amino acids of fibronectin type 3 domain.

<p>(A) Schematic diagram of C-terminal of AGS8 (AGS8-C) and the deleted mutant of AGS8-C (delta AGS8-C) lacking the first 45 amino acids of fibronectin type 3 domain, that are A¹⁴⁹⁴ to R¹⁵³⁸ of rat AGS8 (ABB82299). (B) Bioactivity of AGS8C and delta AGS8C on G-protein activation. The yeast strain expressing human Gαs was transformed with AGS8-C and delta AGS8-C in the pYES2-containing GAL1 promoter. The yeast strain was modified to grow without histidine on activation of G-protein. Induction (+): induction of translation of AGS8 domains by galactose. The representative of 4 independent experiments with similar results.</p

Effect of AGS8-peptide on localization of connexin 43 (CX43) of cultured cardiomyocytes.

<p>(A) Cardiomyocytes were exposed 3 times to 30 min of hypoxia (1% oxygen) intermittent with 30 min of reoxygenation 4 h after (without or with) transfection of AGS8-peptide (1 µM) or FITC-conjugated AGS8-peptide (1 µM). (B) Localization of CX43 in the cardiomyocytes under normoxia and hypoxia. The figures demonstrated in the triple color of CX43 (<i>red</i>, <i>arrow</i>), nuclei (DAPI, <i>blue</i>) and FITC-conjugated AGS8-peptide (<i>green</i>). The representative of 5 independent experiments with similar results. (C) The number of cardiomyocytes expressing CX43 in the cell surface were counted. Please note that ∼ 90% cells were detectable at the point of fixation. Data are represent 170–260 cells from 5 of independent experiments. *, <i>p</i><0.05 vs normoxia group.</p

Uptake of connexin selective fluorescence dye, Lucifer Yellow (LY) to the cardiomyocytes.

<p>Cells were incubated with 1-selective binding and/or incorporation of LY was determined fluorescence in the presence of a connexin hemichannel blocker, 50 µM of Lanthanum which was added 30 min prior to LY. *, <i>p</i><0.05 vs cells in normoxia; <i>n.s.</i>, not statistically significant. N = 4 from 4 independent experiments.</p