Adenosine kinase regulation of cardiomyocyte hypertrophy

Abstract

There is evidence that extracellular adenosine can attenuate cardiac hypertrophy, but the mechanism by which this occurs is not clear. Here we investigated the role of adenosine receptors and adenosine metabolism in attenuation of cardiomyocyte hypertrophy. Phenylephrine (PE) caused hypertrophy of neonatal rat cardiomyocytes with increases of cell surface area, protein synthesis, and atrial natriuretic peptide (ANP) expression. These responses were attenuated by 5 μM 2-chloroadenosine (CADO; adenosine deaminase resistant adenosine analog) or 10 μM adenosine. While antagonism of adenosine receptors partially blocked the reduction of ANP expression produced by CADO, it did not restore cell size or protein synthesis. In support of a role for intracellular adenosine metabolism in regulating hypertrophy, the adenosine kinase (AK) inhibitors iodotubercidin and ABT-702 completely reversed the attenuation of cell size, protein synthesis, and expression of ANP by CADO or ADO. Examination of PE-induced phosphosignaling pathways revealed that CADO treatment did not reduce AKT(Ser⁴⁷³) phosphorylation but did attenuate sustained phosphorylation of Raf(Ser³³⁸) (24-48 h), mTOR(Ser²⁴⁴⁸) (24-48 h), p70S6k(Thr³⁸⁹) (2.5-48 h), and ERK(Thr²⁰²/Tyr²⁰⁴) (48 h). Inhibition of AK restored activation of these enzymes in the presence of CADO. Using dominant negative and constitutively active Raf adenoviruses, we found that Raf activation is necessary and sufficient for PE-induced mTORC1 signaling and cardiomyocyte hypertrophy. CADO treatment still blocked p70S6k(Thr³⁸⁹) phosphorylation and hypertrophy downstream of constitutively active Raf, however, despite a high level phosphorylation of ERK(Thr202/Tyr204) and AKT(Ser⁴⁷³). Reduction of Raf-induced p70S6k(Thr³⁸⁹) phosphorylation and hypertrophy by CADO was reversed by inhibiting AK. Together, these results identify AK as an important mediator of adenosine attenuation of cardiomyocyte hypertrophy, which acts, at least in part, through inhibition of Raf signaling to mTOR/p70S6k.

Fig. 1.

Adenosine kinase (AK) inhibitors block effects of 2-chloroadenosine (CADO) on cell area (A and B), atrial natriuretic peptide (ANP) expression (A and C), and protein synthesis (D). Cells were pretreated for 10 min with 5 μM of 1,3-dipropyl-8-cyclopentylxanthine (DPCPX; A₁ antagonist), ZM 241,385 (A_2A antagonist), MRS 1754 (A_2B antagonist) MRS 1191 (A₃ antagonist), 8-sulfophenyltheophylline (8-SPT; nonselective adenosine receptor antagonist), 0.2 μM iodotubercidin (ITU; AK inhibitor), or 0.2 μM ABT-702 (AK inhibitor), followed by CADO (5 μM) for 10 min and then with 50 μM phenylephrine (PE) for 48 h. Treatment with 1 μM adenosine receptor agonists CPA (A₁ agonist), CGS 21680 (A_2A agonist), 5-(N-ethylcarboxamido)adenosine (NECA; nonselective agonist), or 2-Cl-N ⁶-(3-iodobenzyl)adenosine-5′-N-methyluronamide (2Cl-IB-MECA; A₃ agonist) did not attenuate PE-induced increase in cell area (E). Inhibition of equilibrative nucleoside transporter with S-(4-nitrobenzyl)-6-thioinosine (NBTI; 1 μM ) increased cell area in CADO-treated cells (F). Cell area (B) is the average of 4–8 independent experiments, measuring at least 100 cells per condition. ANP expression (C) is the average ANP expression in at least 100 cells per condition normalized to PE to compare between experiments. [³H]leucine incorporation (D) is the average of 3 experiments, each run in triplicate, and normalized to the PE sample to compare between experiments. For cell area and ANP measurement, cells were fixed and immunostained for α-cardiac myosin heavy chain, ANP, and DNA (DAPI). *P < 0.05, compared with PE. #P < 0.05, compared with PE/CADO. Scale bar = 20 μm.

Fig. 2.

Adenosine attenuation of hypertrophy is blocked by inhibition of AK. Cells were treated with PE (50 μM) and/or adenosine [ADO (1 or 10 μM), pentostatin (PENTO; 1 μM), or ITU (0.2 μM)], and [3-H]leucine incorporation was measured over 48 h in triplicate (A and B). For cell area (C and E) and ANP expression (D and E), adenosine deaminase inhibitors were not included, but media, adenosine, and antagonists were replaced at 24 h. At least 100 cells were measured per condition to obtain average cell area and ANP expression. Scale bar = 30 μM. *P < 0.05, compared with PE treated. #P < 0.05, compared with PE/CADO treated.

Fig. 3.

CADO (5 μM) attenuates sustained phosphorylation of mTOR^Ser2448 and p70S6K^Thr389 and late phosphorylation of ERK^{Thr202/Tyr204} but does not alter AKT^Ser473 in PE (50 μM)-treated cells. Iodotubercidin (ITU; 200 nM) restores phosphorylation of p70S6k^Thr389 and ERK^{Thr202/Tyr204} (A). Cell area increase between 24 and 48 h in PE/CADO-treated cells in response to AK inhibition (ITU; 200 nM) is blocked by inhibition of mammalian target of rapamycin (mTOR; rapamycin; 100 nM) but not inhibition of MEK/ERK signaling (U0126; 10 μM; B). Graphs for cell signaling represent phosphorylation state of indicated proteins relative to GAPDH and normalized to 15-min time point for ERK^{Thr202/Tyr204} or 24-h time point for mTOR^Ser2448, p70S6k^Thr389 or Akt^Ser473 to compare between experiments. *P < 0.05, compared with PE at same time point. #P < 0.05, compared with PE/CADO at same time point. †Significant difference compared with PE/CADO/ITU (B). For cell signaling in A, bars represent average of at least 3 experiments for each time point. For cell area, at least 100 cells were measured per condition.

Fig. 4.

Inhibition of AK with ITU (0.2 μM) restores phosphorylation of Raf ^Ser338, mTOR²⁴⁴⁸, and p70S6k^Thr389 in CADO-treated cells at 24 and 48 h but does not significantly affect AMPK^Thr172 phosphorylation. A and B: graphs represent averages of phosphorylation level from 3–10 experiments, relative to PE-treated samples. *P < 0.05, compared with PE. #P < 0.05, compared with PE/CADO.

Fig. 5.

PE activates mTOR/p70S6k through a Raf-dependent mechanism. Dominant negative Raf (DN-Raf) blocks PE induction of mTOR/p70S6k and ERK (A and C), as well as PE-induced increase in cell area (B and D) and ANP expression per cell (B and E). For immunofluoresence (B), cells were stained for ANP, α-cardiac myosin heavy chain, and DNA (DAPI). There was very little ANP stain in absence of PE and no difference between β-Gal and DN-Raf. For p70S6k^Thr389, graph represents average from 2 independent experiments (n = 3) relative to PE + β-Gal. For cell area, graph represents averages from 2 independent experiments, in which >100 cells were measured per condition. For ANP expression, graph represents average ANP expression measured in ≥100 cells per condition *P < 0.05, compared with PE treated. ND is not determined. Scale bar = 20 μm.

Fig. 6.

Active Raf induces mTOR/p70S6k^Thr389, ERK^{Thr202/Tyr204}, and AKT^Ser473 phosphorylation (A and B) and hypertrophy (C and D), but CADO inhibits p70S6k and hypertrophy through an AK-dependent mechanism. Cells were infected with adenovirus expressing constitutively active Raf or β-Gal for 24 h, followed by CADO or CADO + ITU for an additional 24 h. Cells were fixed and stained for α-cardiac myosin heavy chain, ANP, and DNA (DAPI). *P < 0.05, compared with CA-Raf. #P < 0.05, compared with Raf/CADO. Graphs of p-ERK^{Thr202/Tyr204} and p-p70S6k^Thr389 represent the average of 2 experiments each performed in duplicate (n = 4). For images, scale bar = 20 μm.

Fig. 7.

RNAi knockdown of AK (72 h) results in reduction in AK protein by western blot compared with nontargeting control RNAi-treated cells (A). Immunofluorescent staining of AK is localized in the nucleus in cardiomyocytes, and this staining is lost after 96 h of RNAi treatment (B). RNAi knockdown of AK promotes spontaneous hypertrophy and ANP expression (B, C, D, and E), as well as reduced sensitivity of p70S6k to inhibition by CADO (F). For cell area and ANP expression, cardiomyocytes were transfected with RNAi for 72 h and then treated an additional 48 h with CADO (5 μM). Cells were then fixed and stained for α-cardiac myosin heavy chain, ANP, and DNA (Hoescht). For analysis of p70S6k and mTOR phosphorylation, cell lysates were collected after 24 h of treatment (96 h after RNAi). Graphs represent average of 3 experiments relative to AK RNAi. *P < 0.05, comparing untreated and CADO-treated cells. #P < 0.05, comparing control RNAi-treated cells and AK RNAi-treated cells. Scale bar = 20 μM for B and 40 μM for C.