Mechanistic target of rapamycin complex 2 protects the heart from ischemic damage

Abstract

Background: The mechanistic target of rapamycin (mTOR) comprises 2 structurally distinct multiprotein complexes, mTOR complexes 1 and 2 (mTORC1 and mTORC2). Deregulation of mTOR signaling occurs during and contributes to the severity of myocardial damage from ischemic heart disease. However, the relative roles of mTORC1 versus mTORC2 in the pathogenesis of ischemic damage are unknown.

Methods and results: Combined pharmacological and molecular approaches were used to alter the balance of mTORC1 and mTORC2 signaling in cultured cardiac myocytes and in mouse hearts subjected to conditions that mimic ischemic heart disease. The importance of mTOR signaling in cardiac protection was demonstrated by pharmacological inhibition of both mTORC1 and mTORC2 with Torin1, which led to increased cardiomyocyte apoptosis and tissue damage after myocardial infarction. Predominant mTORC1 signaling mediated by suppression of mTORC2 with Rictor similarly increased cardiomyocyte apoptosis and tissue damage after myocardial infarction. In comparison, preferentially shifting toward mTORC2 signaling by inhibition of mTORC1 with PRAS40 led to decreased cardiomyocyte apoptosis and tissue damage after myocardial infarction.

Conclusions: These results suggest that selectively increasing mTORC2 while concurrently inhibiting mTORC1 signaling is a novel therapeutic approach for the treatment of ischemic heart disease.

Keywords: AKT1S1 protein, human; RICTOR protein, human; TOR serine-threonine kinases.

Figure 1

Decreasing mechanistic target of rapamycin complex 1 (mTORC1) and 2 (mTORC2) activity increases damage after stress. A, Schematic overview of mTOR signaling. B, mTORC1 and mTORC2 are inactivated after treatment with Torin1, as shown by immunoblots. C, Cell death in neonatal rat cardiomyocytes. Challenge with H₂O₂ (50 μmol/L for 4 hours) after mTOR kinase inhibition with Torin1 (50 nmol/L). Torin1 exposure increases apoptosis in response to H₂O₂. *P<0.05 vs vehicle (Veh); #P<0.05 vs H₂O₂ vehicle. D, Torin1 inhibits mTOR kinase activity in vivo, as shown by immunoblots. E, Kaplan–Meier survival curve of vehicle- and Torin1- (5 mg/kg body weight) injected mice. Mortality early after infarction is increased after injection of Torin1. n=4 in the sham groups. F, Torin1 increases infarct size. P<0.05 vs control myocardial infarction (MI). G, Representative confocal scans are shown for terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL), actin, and nuclei (red, green and blue, respectively, in overlays). Bar, 150 μm. Percentage of TUNEL-labeled cells in the left ventricle (LV) of remote area 1 day after MI. *P<0.05 vs control MI. H, Echocardiographic assessment of control or Torin1-injected mice for ejection fraction and LV end-diastolic volume (EDV). *P<0.05 vs sham; #P<0.05 vs MI vehicle. Hemodynamic measurements of ±dP/dt 2 weeks after surgery. *P<0.05 vs sham; #P<0.05 vs MI vehicle. Numbers of mice or of independent experiments per group are indicated in the bar graphs.

Figure 2

Loss of mechanistic target of rapamycin complex 2 (mTORC2) signaling increases cardiomyocyte death in vitro. A, Silencing of Raptor confirmed by immunoblot. B, Bar graphs depicting quantification of Akt phosphorylation and ribosomal S6 protein (RibS6) phosphorylation after Raptor silencing. *P<0.05 vs scramble (Scr); #P<0.05 vs Scr H₂O₂ vehicle (Veh). C, Bar graphs depicting quantitation of Rictor, mTOR, and Raptor expression after Raptor silencing. n=4 independent experiments. D, Cell death in neonatal rat cardiomyocytes. Rictor silencing but not Raptor silencing increases cell death after challenge with H₂O₂ (50 μmol/L for 4 hours). *P<0.05 vs control; #P<0.05 vs Scr H₂O₂. n=5 independent experiments. E, Silencing of Rictor confirmed by immunoblot. n=4 independent experiments. F, Bar graphs depicting quantification of Akt phosphorylation and RibS6 phosphorylation after Rictor silencing. *P<0.05 vs Scr; #P<0.05 vs Scr H₂O₂ vehicle. n=4 independent experiments. G, Histogram depicting quantification of Raptor, mTOR, and Rictor expression after Rictor silencing. n=4 independent experiments.

Figure 3

Loss of mechanistic target of rapamycin complex 2 (mTORC2) signaling increases cardiomyocyte damage after myocardial infarction (MI). A, Silencing of Rictor confirmed by immunoblot. Adeno-associated virus serotype 9 (AAV9)–sh-Rictor hearts exhibit decreased Akt phosphorylation without altered mTORC1 activation. B, Bar graphs depicting quantification of Raptor/mTOR/ Rictor expression. *P<0.05 vs control sham. n=3 per group. C, Bar graphs depicting quantification of ribosomal S6 protein (pRibS6) phosphorylation. n=3 per group. D, Kaplan–Meier survival curve of AAV–sh-control and AAV–sh-Rictor mice. AAV–sh-Rictor increases mortality early after infarction. n=6 in the sham groups. E, Percentage of terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL)–labeled cells in the left ventricle of the remote area 1 day after myocardial infarction (MI).*P<0.05 vs control MI. F, Echocardiographic assessment of AAV–sh-control or AAV–sh-Rictor mice for ejection fraction (EF) and left ventricular end-diastolic volume (LVEDV). *P<0.05 vs sh-control sham; #P<0.05 vs MI sh-Control. G, Silencing of Rictor in myocytes confirmed by confocal microscopy. Representative confocal scans for Rictor, actin, and nuclei (green, red, and blue, respectively, in overlays). Interstitial nonmyocytes express Rictor in AAV–sh-Rictor hearts (arrow), Bar, 150 μm. H, Masson trichrome staining from control and sh-Rictor– treated hearts. Bar graphs depicting quantification of fibrotic area. *P<0.05 vs control sham; #P<0.05 vs MI. Bar, 1mm. Numbers of mice per group are indicated in the bar graphs.

Figure 4

Mechanistic target of rapamycin complex 2 (mTORC2) function is necessary for PRAS40-mediated protection. A, Cell death quantified by flow cytometric detection on H₂O₂ treatment. Torin1 blunts the protective effects of PRAS40. *P<0.05 vs control; $P<0.05 vs control H₂O₂. B, mTOR kinase inhibition after Torin1 (50 nmol/L) confirmed by immunoblot. C, siRNA Akt silencing blunts PRAS40-induced protection. Data are depicted as percentage of dead cells. ***P<0.001 vs control; #P<0.05 vs control H₂O₂. D, Akt1 silencing confirmed by immunoblot. E, siRNA Rictor silencing, but not Raptor silencing, blunts PRA40-induced protection. Data are depicted as percentage of dead cells. ***P<0.001 vs control; #P<0.05 vs control H₂O₂; $$P<0.01 vs control H₂O₂. F, Rictor or Raptor silencing and blunting of mTOR downstream target phosphorylation by immunoblot. Numbers of independent experiments per group are indicated in the bar graphs.

Figure 5

PRAS40 protects against ischemic injury. A, Kaplan–Meier survival analysis of control and PRAS40 animals after myocardial infarction (MI) or sham operation. Masson trichrome staining of control and PRAS40-treated hearts 6 weeks after surgery. n=5 for the sham groups. B, Infarct size measurements 6 weeks after MI. *P<0.05 vs control MI. C, Percentage of terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL)–labeled myocytes in the left ventricle (LV) in the remote area 2 days after MI. *P<0.05 vs control MI. D, Weekly echocardiographic assessment of control or PRAS40 sham and MI hearts for ejection fraction and LV enddiastolic volume (LVEDV). *P<0.05 vs control MI; n=14 to 15 for the MI group and n=5 to 6 for the sham group. E, In vivo hemodynamic measurements of ±dP/dt and LV end-diastolic pressure (LVEDP) 6 weeks after surgery. F, Ratio of heart weight to body weight (HW/BW) in control and PRAS40 mice 6 weeks after sham and MI surgery. G, Cardiomyocyte area (CSA) in control and PRAS40 mice 6 weeks after sham or MI surgery (*P<0.01 vs control sham; #P<0.05 vs control MI. H, Nppa and Nppb transcription in hearts of mice of the indicated group 6 weeks after sham or MI (*P<0.01 vs control sham; #P<0.05 vs control MI). Error bars indicate mean±SEM. I, Masson trichrome staining from control and PRAS40-treated hearts. Bar graphs depicting quantification of fibrotic area. *P<0.05 vs control sham. #P<0.05 vs MI. Bar, 1 mm. J, Collagen1 transcription in hearts of mice of the indicated group 6 weeks after sham or MI. *P<0.01 vs control sham; #P<0.05 vs control MI. Error bars indicate mean±SEM. Number of mice per group is indicated in the bar.

Figure 6

PRAS40 promotes protective mechanistic target of rapamycin complex 2 (mTORC2) signaling in vivo. A, Heart lysates for indicated proteins 2 days after myocardial infarction (MI) assessed by immunoblot. Akt phosphorylation quantification histogram. P<0.01 vs control sham; #P<0.05 vs control MI. B, Heart lysates for indicated proteins 2 days after MI assessed by immunoblot. Ribosomal S6 protein (RibS6) phosphorylation quantification by histogram. *P<0.01 vs control sham; #P<0.05 vs control MI. C, Paraffin-embedded sections from control hearts and PRAS40-treated hearts 2 days after MI stained for pAkt⁴⁷³ (red), actin (green), and nuclei (blue) and assessed by confocal microscopy. D, Sections of control hearts and PRAS40-treated hearts 2 days after MI stained for pRibS6 (red), actin (green), and nuclei (blue) and assessed by confocal microscopy. Bar, 150 μm. E, Heart lysates assessed for indicated protein expression 6 weeks after surgery by immunoblot. F, PRAS40 overexpression increases Akt phosphorylation in myocytes by confocal microscopy of myocardial sections at 6 weeks after surgery. Bar, 50 μm. Number of mice per group is indicated in the bar.

Figure 7

Cardioprotection by PRAS40 is mechanistic target of rapamycin complex 2 (mTORC2) dependent in vivo. A, Ejection fraction (EF) and left ventricular end-diastolic volume (LVEDV) in PI3K^−/− mice before and after myocardial infarction (MI) assessed by echocardiography. B, EF and LVEDV in Akt1^−/− mice before and after MI assessed by echocardiography. C, EF and LVEDV in mice injected with adeno-associated virus serotype 9 (AAV9)–sh-control, AAV–sh-Rictor, AAV-PRAS40, or AAV–sh-Rictor and AAV-PRAS40 and assessed by echocardiography. *P<0.05 vs sh-control; #P<0.05 vs sh-control. D, Ratio of heart weight to body weight (HW/BW) in mice 2 weeks after MI surgery. *P<0.05 vs sh-control. E, Percentage of terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL)– labeled myocytes in the LV in the remote area 1 day after MI. n=3 per group. *P<0.05 vs control MI; #P<0.05 vs sh-control. Bar, 150 μm. Number of mice per group is indicated in the bar.

Figure 8

Model for PRAS40-mediated cardioprotection. mechanistic target of rapamycin (mTOR) kinase inhibitors or impairment of mTOR complex 2 (mTORC2) function worsens cardiac function after myocardial infarction. PRAS40 blocks mTORC1 in myocytes and diverts toward mTORC2 function, increasing Akt activation and leading to increased cellular survival after infarction. IRS-1 indicates insulin receptor substrate-1.