Activator of G Protein Signaling 8 (AGS8) Is Required for Hypoxia-induced Apoptosis of Cardiomyocytes

Abstract

Ischemic injury of the heart is associated with activation of multiple signal transduction systems including the heterotrimeric G-protein system. Here, we report a role of the ischemia-inducible regulator of G␤␥ subunit, AGS8, in survival of cardiomyocytes under hypoxia. Cultured rat neonatal cardiomyocytes (NCM) were exposed to hypoxia or hypoxia/ reoxygenation following transfection of AGS8siRNA or pcDNA::AGS8. Hypoxia-induced apoptosis of NCM was completely blocked by AGS8siRNA, whereas overexpression of AGS8 increased apoptosis. AGS8 formed complexes with G-proteins and channel protein connexin 43 (CX43), which regulates the permeability of small molecules under hypoxic stress. AGS8 initiated CX43 phosphorylation in a G␤␥-dependent manner by providing a scaffold composed of G␤␥ and CX43. AGS8siRNA blocked internalization of CX43 following exposure of NCM to repetitive hypoxia; however it did not influence epidermal growth factor-mediated internalization of CX43. The decreased dye flux through CX43 that occurred with hypoxic stress was also prevented by AGS8siRNA. Interestingly, the G␤␥ inhibitor Gallein mimicked the effect of AGS8 knockdown on both the CX43 internalization and the changes in cell permeability elicited by hypoxic stress. These data indicate that AGS8 is required for hypoxia-induced apoptosis of NCM, and that AGS8-G␤␥ signal input increased the sensitivity of cells to hypoxic stress by influencing CX43 regulation and associated cell permeability. Under hypoxic stress, this unrecognized response program plays a critical role in the fate of NCM. G-protein-coupled receptors (GPCRs)4 are signaling proteins on the cell surface responsible for mediating various ligands, such as hormones and neurotransmitters. Activation of cell surface GPCRs initiates nucleotide exchange on G␣ subunits, which leads to a conformational change of G␣␤␥ and subsequent transduction of signals to various intracellular effector molecules (1-3). In addition to such established signaling pathways, recent studies indicate the existence of a novel class of regulatory proteins for heterotrimeric G-proteins. These regulatory proteins may provide alternative signal processing via G␣␤␥, G␣, or G␤␥ subunits distinct from typical GPCR pathways, and identifying these mechanisms may uncover unrecognized roles of G-proteins beyond simple transducers of signals from GPCRs (4 -6).During the alteration of signaling pathways in disease states, such regulatory proteins may be involved in adaptation programs of cells to maintain homeostasis (7-11). Genetic modification of regulatory proteins for G-proteins leads to the development of cardiovascular dysfunction in mice including hypertension, maladaptive response to pressure overload, or altered baroreceptor reflex (9, 12, 13). Thus, such regulatory accessory proteins may be involved in the development of disease via either regulating GPCR-initiated signals or by undefined, alternative G-protein signaling pathways operating independent of the receptor.In our efforts to adaptation-specific sig...

Identification of Transcription Factor E3 (TFE3) as a Receptor-independent Activator of Gα16

“…The conditions and duplex eliciting the most effective reduction in GNA15 and TFE3 were determined in a series of preliminary experiments. Cardiomyocytes were prepared from the hearts of 1-3-day-old Wistar rats as described previously (24). Approximately 24 h after preparation, neonatal cardiomyocytes at 4.0 ϫ 10 5 cells in 35-mm plates were transfected with siRNA using Lipofectamine 2000 according to the manufacturer's instructions.…”

“…Immunoblotting and data analysis were performed as described previously (18,24). The luminescence images captured with an image analyzer (LAS-3000, Fujifilm, Tokyo, Japan) were quantified using Image Gauge 3.4 (Fujifilm).…”

“…The samples were incubated with 25 l of 50% Sepharose-G for 1 h at 4°C, and the pellets were washed three times with immunoprecipitation buffer. Proteins were eluted in 30 l of 2ϫ Laemmli buffer and resolved by SDS-PAGE (24).…”

“…RNA isolation, cDNA synthesis, and real time PCR analysis were performed as described previously (24). The primers for RT-PCR were as follows: mouse MITF: forward, 5Ј-ACTTTC-CCTTATCCCATCCACC-3Ј; reverse, 5Ј-TGAGATCCAGA-GTTGTCGTACA-3Ј; mouse TFE3: forward, 5Ј-TGCGTCAG-CAGCTTATGAGG-3Ј; reverse, 5Ј-AGACACGCCAATCAC-AGAGAT-3Ј; mouse TFEB: forward, 5Ј-CCACCCCAGCCAT-CAACAC-3Ј; reverse, 5Ј-CAGACAGATACTCCCGAAC-CTT-3Ј; mouse GNA15: forward, 5Ј-CGCCAGAATCGACC-AGGAG-3Ј; reverse, 5Ј-GTAGCCCACACCGTGAATGA-3Ј; mouse claudin 14: forward, 5Ј-GCATGGTGGGAACGCT-CAT-3Ј; reverse, 5Ј-CCACAGTCCCTTCAGGTAGGA-3Ј; human claudin 14: forward, 5Ј-CAAACACCGCACCTGC-CTA-3Ј; reverse, 5Ј-CACGTAGTCGTTCAGCCTGT-3Ј; rat GNA15: forward, 5Ј-CAGGAGAACCGTATGAAGGAGA-GTC-3Ј; reverse, 5Ј-CAGGATGTCTGTCTTGTTGAGGAAG-3Ј; rat TFE3: forward, 5Ј-TGTTCGTGCTGTTGGAAGAGC-3Ј; reverse, 5Ј-GGGATAGAGGCTGGCTTTTGAG-3Ј; rat claudin 14: forward, 5Ј-TCATCACTACTATCCTGCCGCAC-3Ј; reverse, 5Ј-ACACACTCCATCCACAGTCCCTTC-3Ј; and 18 S: forward, 5Ј-GTAACCCGTTGAACCCCATT-3Ј; reverse, 5Ј-CCATCCAATCGGTAGTAGCG-3Ј.…”

“…AGS8 was up-regulated in cardiomyocytes in response to transient hypoxia and regulated G␤␥ signaling. Indeed, AGS8 played a key role in apoptosis of cardiomyocytes induced by hypoxic stress via G␤␥ and the channel protein connexin 43 (24). These findings prompted us to investigate the presence of putative AGS proteins in other models of cardiovascular diseases.…”