Rapamycin treatment augments both protein ubiquitination and Akt activation in pressure-overloaded rat myocardium

Abstract

Ubiquitin-mediated protein degradation is necessary for both increased ventricular mass and survival signaling for compensated hypertrophy in pressure-overloaded (PO) myocardium. Another molecular keystone involved in the hypertrophic growth process is the mammalian target of rapamycin (mTOR), which forms two distinct functional complexes: mTORC1 that activates p70S6 kinase-1 to enhance protein synthesis and mTORC2 that activates Akt to promote cell survival. Independent studies in animal models show that rapamycin treatment that alters mTOR complexes also reduces hypertrophic growth and increases lifespan by an unknown mechanism. We tested whether the ubiquitin-mediated regulation of growth and survival in hypertrophic myocardium is linked to the mTOR pathway. For in vivo studies, right ventricle PO in rats was conducted by pulmonary artery banding; the normally loaded left ventricle served as an internal control. Rapamycin (0.75 mg/kg per day) or vehicle alone was administered intraperitoneally for 3 days or 2 wk. Immunoblot and immunofluorescence imaging showed that the level of ubiquitylated proteins in cardiomyocytes that increased following 48 h of PO was enhanced by rapamycin. Rapamycin pretreatment also significantly increased PO-induced Akt phosphorylation at S473, a finding confirmed in cardiomyocytes in vitro to be downstream of mTORC2. Analysis of prosurvival signaling in vivo showed that rapamycin increased PO-induced degradation of phosphorylated inhibitor of κB, enhanced expression of cellular inhibitor of apoptosis protein 1, and decreased active caspase-3. Long-term rapamycin treatment in 2-wk PO myocardium blunted hypertrophy, improved contractile function, and reduced caspase-3 and calpain activation. These data indicate potential cardioprotective benefits of rapamycin in PO hypertrophy.

Fig. 1.

Localized ubiquitination (Ub) at the intercalated disc is enhanced by rapamycin treatment during pressure overload (PO). A: Triton-insoluble proteins were normalized to actin and analyzed by immunoblotting with anti-ubiquitin antibody. The summary data for quantification conducted using ImageJ is represented in the graph as means ± SE. *P < 0.05 vs. control; #P < 0.05 vs. PO vehicle. (n = 4). RV, right ventricle; LV, left ventricle; Rapa, rapamycin; Cont, control. B: confocal microscopic analysis was performed on 10-μm sections of PO ventricles from vehicle (top) or rapamycin-treated (bottom) rats stained with anti-N-cadherin (green) and anti-ubiquitin (red). The higher-magnification images below each treatment group show anti-N-cadherin (green) and anti-ubiquitin (red) on the left and anti-α-actinin (green) and anti-ubiquitin (red) on the right. Scale = 10 μm.

Fig. 2.

Rapamycin augments Akt activity induced by PO in vivo. Triton-soluble proteins were normalized to GAPDH and analyzed by immunoblotting with anti-pS473-Akt, Akt, and p389-p70S6 kinase-1 (S6K1) antibodies. Control LV and RV samples for pT389-S6K1 were run on the same gel, but they were noncontiguous as indicated by the vertical line. The summary data for quantification conducted using ImageJ for pS473-Akt are represented in the graph as means ± SE. *P < 0.05 vs. control; #P < 0.05 vs. PO vehicle (n = 4).

Fig. 3.

Rapamycin augments PO-induced changes in inhibitor of κB (IκB). Ventricles of control and PO rats with or without rapamycin were lysed in Triton buffer, and soluble proteins were obtained by centrifugation. Triton-soluble proteins were normalized to GAPDH and analyzed by immunoblotting with anti-IκB and pIκB antibodies. The summary data of quantification conducted using ImageJ are represented in both graphs as means ± SE. *P < 0.05 vs. control; #P < 0.05 vs. PO vehicle (n = 4).

Fig. 4.

Rapamycin alters PO-induced changes in cIAP1 and caspase-3. Ventricles of control and PO rats with and without rapamycin were lysed in Triton buffer, and insoluble and soluble proteins were separated by centrifugation. A: insoluble and soluble proteins were normalized to actin and GAPDH, respectively, and analyzed by immunoblotting with anti-cIAP1 antibody. The summary data of quantification conducted using ImageJ are represented in the graph as means ± SE. *P < 0.05 vs. control; #P < 0.05 vs. PO vehicle, (n ≥ 4/group). B: insoluble proteins, normalized to actin, were analyzed by immunoblotting with anti-procaspase-3 (full-length caspase-3) and anti-active caspase-3 (cleaved caspase-3) antibodies. The active caspase-3 antibody recognizes cleaved active fragments of caspase-3 (17–20 kDa) that are indicated by the bracket. The summary data of quantification conducted using ImageJ are represented in the graph as means ± SEM. *P < 0.05 vs. control; #P < 0.05 vs. PO vehicle, (n = 4). All caspase-3 fragments were used for calculations of active caspase-3.

Fig. 5.

Effect of long-term (2 wk) rapamycin treatment in PO myocardium. Rats that underwent sham surgery or PO were treated with a daily dose of vehicle alone or rapamycin for 14 days. The animals were euthanized, and the hearts were used for the isolation of either RV papillary muscle or free wall. A: shortening percent was determined from the isolated RV papillary muscle. *P < 0.05 vs. corresponding control; #P < 0.05 vs. control + vehicle. PAB, pulmonary artery banding. B: RV tissue sections were fresh frozen and cryosectioned (12-μm sections). The sections were stained for active caspase-3 (red), μ-calpain (green), and DAPI (blue) and analyzed by using confocal microscopy.

Fig. 6.

Rapamycin augments Akt activation in agonist-stimulated adult rat cardiomyocytes via mammalian target of rapamycin (mTOR)-mediated signaling. Triton-soluble proteins were extracted from isolated rat cardiomyocytes and mTOR complex 1 (mTORC1) and mTORC2 activation was analyzed though immunoblotting with anti-pT389-S6K1 and anti-pS473-Akt antibodies, respectively. Vehicle-treated cells were controls in each experiment. Total protein was normalized to GAPDH. A: cardiomyocytes were pretreated with rapamycin (2 nM, long is L = 24 h, short is S = 30 min) before insulin treatment (100 nM, 30 min). B: cardiomyocytes were supplemented with insulin (100 nM) and treated with rapamycin (2 nM) for the indicated times. C: cardiomyocytes were pretreated with rapamycin (2 nM, 30 min) before 1-h stimulation with either phenylephrine (PE) (100 μM) or endothelin (ET) (400 nM). D: cardiomyocytes were exposed to β-galactosidase (β-gal) 200 multiplicity of infection (MOI) and rapamycin-resistant S6K1 adenovirus (RR-S6K1) at 100 and 200 MOI for 48 h. Cells were then pretreated with rapamycin (2 nM, 30 min) before insulin treatment (100 nM, 30 min). Soluble proteins were analyzed by immunoblotting with anti-pS473-Akt, and insoluble proteins were analyzed with anti-pS235/S236-S6 Protein antibody. E: cardiomyocytes were pretreated with torin (100 nM, 1 h) then treated with rapamycin (2 nM, 30 min) before insulin stimulation (100 nM, 30 min). Results were confirmed by performing 2 additional experiments.