The mechanistic target of rapamycin complex-1 (mTORC1) coordinates regulation of growth, metabolism, protein synthesis and autophagy1. Its hyperactivation contributes to disease in numerous organs, including the heart1,2, although broad inhibition of mTORC1 risks interference with its homeostatic roles. Tuberin (TSC2) is a GTPase-activating protein and prominent intrinsic regulator of mTORC1 that acts through modulation of RHEB (Ras homologue enriched in brain). TSC2 constitutively inhibits mTORC1; however, this activity is modified by phosphorylation from multiple signalling kinases that in turn inhibits (AMPK and GSK-3β) or stimulates (AKT, ERK and RSK-1) mTORC1 activity3,4,5,6,7,8,9. Each kinase requires engagement of multiple serines, impeding analysis of their role in vivo. Here we show that phosphorylation or gain- or loss-of-function mutations at either of two adjacent serine residues in TSC2 (S1365 and S1366 in mice; S1364 and S1365 in humans) can bidirectionally control mTORC1 activity stimulated by growth factors or haemodynamic stress, and consequently modulate cell growth and autophagy. However, basal mTORC1 activity remains unchanged. In the heart, or in isolated cardiomyocytes or fibroblasts, protein kinase G1 (PKG1) phosphorylates these TSC2 sites. PKG1 is a primary effector of nitric oxide and natriuretic peptide signalling, and protects against heart disease10,11,12,13. Suppression of hypertrophy and stimulation of autophagy in cardiomyocytes by PKG1 requires TSC2 phosphorylation. Homozygous knock-in mice that express a phosphorylation-silencing mutation in TSC2 (TSC2(S1365A)) develop worse heart disease and have higher mortality after sustained pressure overload of the heart, owing to mTORC1 hyperactivity that cannot be rescued by PKG1 stimulation. However, cardiac disease is reduced and survival of heterozygote Tsc2S1365A knock-in mice subjected to the same stress is improved by PKG1 activation or expression of a phosphorylation-mimicking mutation (TSC2(S1365E)). Resting mTORC1 activity is not altered in either knock-in model. Therefore, TSC2 phosphorylation is both required and sufficient for PKG1-mediated cardiac protection against pressure overload. The serine residues identified here provide a genetic tool for bidirectional regulation of the amplitude of stress-stimulated mTORC1 activity.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

Data availability

The authors declare that the data supporting the findings of this study are available within the paper and the Supplementary Information. Numerical values corresponding to figures that describe the results from in vivo model studies are provided as separate Source Data for Figs. 1f–h, 2a, 3d, 4c, e and Extended Data Fig. 1a. Other source data related to the study are available from the corresponding author upon reasonable request. Any reagents developed for this study, including novel plasmids, viral vectors and the Tsc2 knock-in mouse models can be made available upon direct request to the corresponding author.

Additional information

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


  1. 1.

    Saxton, R. A. & Sabatini, D. M. mTOR signaling in growth, metabolism, and disease. Cell 168, 960–976 (2017).

  2. 2.

    Sciarretta, S., Forte, M., Frati, G. & Sadoshima, J. New insights into the role of mTOR signaling in the cardiovascular system. Circ. Res. 122, 489–505 (2018).

  3. 3.

    Zhang, Y. et al. Coordinated regulation of protein synthesis and degradation by mTORC1. Nature 513, 440–443 (2014).

  4. 4.

    Ma, L., Chen, Z., Erdjument-Bromage, H., Tempst, P. & Pandolfi, P. P. Phosphorylation and functional inactivation of TSC2 by Erk implications for tuberous sclerosis and cancer pathogenesis. Cell 121, 179–193 (2005).

  5. 5.

    Potter, C. J., Pedraza, L. G. & Xu, T. Akt regulates growth by directly phosphorylating Tsc2. Nat. Cell Biol. 4, 658–665 (2002).

  6. 6.

    Roux, P. P., Ballif, B. A., Anjum, R., Gygi, S. P. & Blenis, J. Tumor-promoting phorbol esters and activated Ras inactivate the tuberous sclerosis tumor suppressor complex via p90 ribosomal S6 kinase. Proc. Natl Acad. Sci. USA 101, 13489–13494 (2004).

  7. 7.

    Menon, S. et al. Spatial control of the TSC complex integrates insulin and nutrient regulation of mTORC1 at the lysosome. Cell 156, 771–785 (2014).

  8. 8.

    Inoki, K., Zhu, T. & Guan, K. L. TSC2 mediates cellular energy response to control cell growth and survival. Cell 115, 577–590 (2003).

  9. 9.

    Inoki, K. et al. TSC2 integrates Wnt and energy signals via a coordinated phosphorylation by AMPK and GSK3 to regulate cell growth. Cell 126, 955–968 (2006).

  10. 10.

    Lee, D. I. et al. Phosphodiesterase 9A controls nitric-oxide-independent cGMP and hypertrophic heart disease. Nature 519, 472–476 (2015).

  11. 11.

    Kim, G. E. & Kass, D. A. Cardiac phosphodiesterases and their modulation for treating heart disease. Handb. Exp. Pharmacol. 243, 249–269 (2016).

  12. 12.

    Takimoto, E. et al. Chronic inhibition of cyclic GMP phosphodiesterase 5A prevents and reverses cardiac hypertrophy. Nat. Med. 11, 214–222 (2005).

  13. 13.

    Kinoshita, H. et al. Inhibition of TRPC6 channel activity contributes to the antihypertrophic effects of natriuretic peptides–guanylyl cyclase-A signaling in the heart. Circ. Res. 106, 1849–1860 (2010).

  14. 14.

    Kim, J., Kundu, M., Viollet, B. & Guan, K. L. AMPK and mTOR regulate autophagy through direct phosphorylation of Ulk1. Nat. Cell Biol. 13, 132–141 (2011).

  15. 15.

    Hariharan, N., Zhai, P. & Sadoshima, J. Oxidative stress stimulates autophagic flux during ischemia/reperfusion. Antioxid. Redox Signal. 14, 2179–2190 (2011).

  16. 16.

    Ballif, B. A. et al. Quantitative phosphorylation profiling of the ERK/p90 ribosomal S6 kinase-signaling cassette and its targets, the tuberous sclerosis tumor suppressors. Proc. Natl Acad. Sci. USA 102, 667–672 (2005).

  17. 17.

    Mertins, P. et al. Proteogenomics connects somatic mutations to signalling in breast cancer. Nature 534, 55–62 (2016).

  18. 18.

    Wong, A., Zhang, Y. W., Jeschke, G. R., Turk, B. E. & Rudnick, G. Cyclic GMP-dependent stimulation of serotonin transport does not involve direct transporter phosphorylation by cGMP-dependent protein kinase. J. Biol. Chem. 287, 36051–36058 (2012).

  19. 19.

    Allen, J. J. et al. A semisynthetic epitope for kinase substrates. Nat. Methods 4, 511–516 (2007).

  20. 20.

    Taneike, M. et al. mTOR hyperactivation by ablation of tuberous sclerosis complex 2 in the mouse heart induces cardiac dysfunction with the increased number of small mitochondria mediated through the down-regulation of autophagy. PLoS ONE 11, e0152628 (2016).

  21. 21.

    Zhang, D. et al. mTORC1 regulates cardiac function and myocyte survival through 4E-BP1 inhibition in mice. J. Clin. Invest. 120, 2805–2816 (2010).

  22. 22.

    Shende, P. et al. Cardiac raptor ablation impairs adaptive hypertrophy, alters metabolic gene expression, and causes heart failure in mice. Circulation 123, 1073–1082 (2011).

  23. 23.

    Moschella, P. C., Rao, V. U., McDermott, P. J. & Kuppuswamy, D. Regulation of mTOR and S6K1 activation by the nPKC isoforms, PKCε and PKCδ, in adult cardiac muscle cells. J. Mol. Cell. Cardiol. 43, 754–766 (2007).

  24. 24.

    Fonseca, B. D. et al. Pharmacological and genetic evaluation of proposed roles of mitogen-activated protein kinase/extracellular signal-regulated kinase kinase (MEK), extracellular signal-regulated kinase (ERK), and p90RSK in the control of mTORC1 protein signaling by phorbol esters. J. Biol. Chem. 286, 27111–27122 (2011).

  25. 25.

    Ranganathan, V., Wahlin, K., Maruotti, J. & Zack, D. J. Expansion of the CRISPR–Cas9 genome targeting space through the use of H1 promoter-expressed guide RNAs. Nat. Commun. 5, 4516 (2014).

  26. 26.

    Ran, F. A. et al. Genome engineering using the CRISPR–Cas9 system. Nat. Protoc. 8, 2281–2308 (2013).

  27. 27.

    Zhang, H. et al. Loss of Tsc1/Tsc2 activates mTOR and disrupts PI3K–Akt signaling through downregulation of PDGFR. J. Clin. Invest. 112, 1223–1233 (2003).

  28. 28.

    Nakamura, T. et al. Prevention of PKG1α oxidation augments cardioprotection in the stressed heart. J. Clin. Invest. 125, 2468–2472 (2015).

  29. 29.

    Scotcher, J. et al. Disulfide-activated protein kinase G Iα regulates cardiac diastolic relaxation and fine-tunes the Frank–Starling response. Nat. Commun. 7, 13187 (2016).

Download references


This study was supported by National Institutes of Health (NIH) National Heart Lung and Blood Institute grants HL-135827, HL-119012, HL089297, T32-HL-07227 (D.A.K.), HHSN268201000032C (J.E.V.E. and D.A.K.), F31-HL134196 (K.M.K.-S.), F31-HL143905 (B.L.D.-E.), American Heart Association Post-Doctoral Fellowships (M.J.R., D.I.L. and T.N.), Deutsche Forschungsgemeinschaft OE 688/1-1 (C.U.O.), Fondation Leducq TransAtlantic Network of Excellence, and an Abraham and Virginia Weiss Professorship (D.A.K.), an Erika J. Glazer Endowed Chair in Women’s Heart Health (J.E.V.E.) and the Barbra Streisand Women’s Heart Center (J.E.V.E.), R01AI077610 and R01AI091481 (J.D.P.), and the Bloomberg~Kimmel Institute for Cancer Immunotherapy (J.D.P.). We thank P. Eaton for providing plasmid constructs expressing PKG1α(WT) and PKG1α(M438G), J. Sadoshima for providing the LC3-II–GFP–RFP reporter-expressing adenovirus, B. Manning for the DNA construct of wild-type human TSC2 and J. T. Kass for assisting with protein kinase bioinformatics analyses.

Author information


  1. Division of Cardiology, Department of Medicine, The Johns Hopkins Medical Institutions, Baltimore, MD, USA

    • Mark J. Ranek
    • , Kristen M. Kokkonen-Simon
    • , Anna Chen
    • , Miguel Pinilla Vera
    • , Christian U. Oeing
    • , Taishi Nakamura
    • , Guangshuo Zhu
    • , Djahida Bedja
    • , Masayuki Sasaki
    • , Dong Ik Lee
    •  & David A. Kass
  2. Department of Pharmacology and Molecular Sciences, Johns Hopkins University, Baltimore, MD, USA

    • Brittany L. Dunkerly-Eyring
    •  & David A. Kass
  3. Bloomberg~Kimmel Institute for Cancer Immunotherapy, Sidney-Kimmel Comprehensive Cancer Research Center, Department of Oncology, Johns Hopkins University School of Medicine, Baltimore, MD, USA

    • Chirag H. Patel
    •  & Jonathan D. Powell
  4. The Smidt Heart Institute and Advanced Clinical Biosystems Research Institute, Cedars Sinai Medical Center, Los Angeles, CA, USA

    • Ronald J. Holewinski
    •  & Jennifer E. Van Eyk


  1. Search for Mark J. Ranek in:

  2. Search for Kristen M. Kokkonen-Simon in:

  3. Search for Anna Chen in:

  4. Search for Brittany L. Dunkerly-Eyring in:

  5. Search for Miguel Pinilla Vera in:

  6. Search for Christian U. Oeing in:

  7. Search for Chirag H. Patel in:

  8. Search for Taishi Nakamura in:

  9. Search for Guangshuo Zhu in:

  10. Search for Djahida Bedja in:

  11. Search for Masayuki Sasaki in:

  12. Search for Ronald J. Holewinski in:

  13. Search for Jennifer E. Van Eyk in:

  14. Search for Jonathan D. Powell in:

  15. Search for Dong Ik Lee in:

  16. Search for David A. Kass in:


M.J.R. performed most of the study, spearheaded the initial identification of the TSC2 modification, and organized and analysed most of the data. K.M.K.-S. had a key role in designing and developing both knock-in mouse lines and gene sequencing analysis. A.C. performed a number of cellular/molecular assays for the study. B.L.D.-E. performed radiolabelled and previously developed kinase assays19, analysed data and identified dual-site regulation. M.P.V. performed AMPK and mTORC2 assays and analysis. C.U.O. performed cardiomyocyte stimulation/autophagy and mTORC1 studies and analysis. C.H.P. developed TSC2 knockout HEK293 cell lines and contributed molecular signalling data. T.N. identified the mTORC1–PKG1 interaction and assisted with initial pressure-overload model studies. G.Z. performed all surgeries to induce pressure overload in mice. D.B. performed and analysed all of the echocardiographic data. M.S. generated numerous plasmids and viral vectors used for the study. R.J.H. performed the PKG1-kinome proteomics analysis. J.E.V.E. supervised the proteomics study, and provided grant support, assistance with project development and the preparation of the manuscript. J.D.P. contributed insights regarding mTORC1–TSC2 signalling, supervised C.H.P. and helped with the preparation of the manuscript. D.I.L. provided human data analysis and assay assistance to many in the study. D.A.K. conceived and directed the overall study, provided the majority of grant support for the research, provided scientific input throughout its development, substantially contributed to data presentation and analysis, and was responsible for the manuscript.

Competing interests

D.A.K., J.D.P., M.J.R., K.M.K.-S. and C.H.P. are co-inventors on a patent application (PCT: 448070145WO1) that was filed in July 2018 (provisional filed in June 2017). The patent relates to the use of TSC2(S1365/S1364) modifications for immunological applications. D.A.K., J.D.P. and M.J.R. are co-founders and shareholders of Meta-T Cellular, a start-up company that aims to develop applications of this intellectual property for immune therapy.

Corresponding author

Correspondence to David A. Kass.

Extended data figures and tables

  1. Extended Data Fig. 1 Both mTORC1 inhibition (everolimus) and PKG1 activation (sildenafil) prevent pathological heart growth, dysfunction, hypertrophic gene expression, mTORC1 activation and myocardial protein aggregation.

    a, Heart weight/tibial length, lung weight/tibial length, cardiac ejection fraction, mRNA expression of Nppa, Nppb and Rcan1 (encoding A- and B-type natriuretic peptide and regulator of calcineurin 1, respectively, and each normalized to Gapdh) from the same study that generated the data shown in Fig. 1a, b. Mice were  subjected to 6 weeks of pressure overload (PO) from trans-aortic constriction or to sham surgery, and pressure-overloaded mice were further treated with vehicle, sildenafil (200 mg kg−1 day−1) or everolimus (10 mg kg−1 day−1) starting 1 week after pressure-overload surgery. Data are mean ± s.e.m.; n = 6 biologically independent experiments, one-way ANOVA with Tukey multiple comparisons test, P ≤ 6 × 10−5 versus the other three groups, §P = 1 × 10−6 versus sham, P = 0.007 versus pressure overload and sildenafil, P = 0.002 versus pressure overload and everolimus; #P = 0.02, P < 0.007, P = 0.02 versus Sham; *P ≤ 0.0005 versus all other groups. b, c, Summary analysis for immunoblots displayed in Fig. 1a, b. Data are mean ± s.e.m.; same 6 biologically independent experiments as in a. One-way ANOVA with Tukey multiple comparisons test, P < 1 × 10−6, ††P < 1 × 10−5 versus the other three groups, #P < 1 × 10−6 versus sham, P =  0.0003 versus pressure overload and vehicle, *P = 0.002 versus sham. d, Filter trap assay from myocardium obtained from same mouse experiment, with membranes probed for ubiquitin and α-tubulin. n = 4 biologically independent experiments, mean ± s.e.m., one-way ANOVA with Tukey multiple comparisons test, P = 1 × 10−6 versus other groups, #P = 0.0002, P = 0.001 versus sham.

  2. Extended Data Fig. 2 PKG1 activation enhances autophagic flux and this is required for anti-hypertrophic efficacy and leads to TSC2 phosphorylation at S1364 (human; 1365 mouse; 1366 rat).

    a, Immunoblot of LC3-II in NRCMs with and without BFA treatment to block autophagy by preventing lysosomal proteolysis. The relative increase in LC3-II expression without versus with BFA treatment indexes autophagic flux. An example blot is shown on the left, summary data on the right. n = 4 biologically independent experiments; data are mean ± s.d., two-way ANOVA with Tukey multiple pairwise comparisons test, P < 0.0001 for interaction between ±BFA and drug treatment; *P = 1.5 × 10−5, #P < 1 × 10−6, §P = 0.013,  P = 0.0002 for within group comparison ±BFA; P < 1 × 10−6 versus each of the other groups with BFA. b, Effect of siRNA gene knockdown of Atg5 (siATG5) versus scrambled control (Scr-siRNA) on Nppb gene expression in NRCMs treated with ET1 with or without sildenafil. n = 6 biologically independent samples, data are mean ± s.d., one-way ANOVA and Tukey multiple comparisons test, P < 1 × 10−6 versus three groups in Scr-siRNA, #P < 1 × 10−6 versus ET1 and sildenafil (Sil) in siATG5 group. c, Mass spectrometry identifies Tsc2 S1366 in rat (equivalent to human S1364 and mouse S1365) as a phosphorylation target of PKG1. Adult rat ventricular myocytes were exposed to cGMP to stimulate PKG1 activity and mass spectrometry was performed on three independent replicates. d, Summary of immunoblot experiment in Fig. 1e. MEFs treated with 8-bromo-cGMP with and without the PKG1 inhibitor DT3. n = 6 biologically independent samples, data are mean ± s.d., one-way ANOVA with post hoc Tukey test: P = 0.00004 versus vehicle, P = 0.0004 versus cGMP. e, Immunoblot for TSC2 (antibody that recognizes the C terminus) in cardiomyocytes expressing native protein, or transduced with TSC2(WT), TSC2(S1365A) or TSC2(S1365E) using plasmid vector transfection. Expression levels were similar with each plasmid at twice the level of non-transduced cells. n = 4 biologically independent samples, data are mean ± s.d., one-way ANOVA with post hoc Tukey test, *P = 0.0004 versus control.

  3. Extended Data Fig. 3 PKG1 is activated by ET1 in rat cardiomyocytes and results in TSC2 phosphorylation detected by a human TSC2(S1364) antibody in cells expressing human TSC2(WT) but not human TSC2(S1364A) or TSC2(S1365A); this phosphorylation occurs by direct modification of TSC2 by PKG1α.

    a, PKG1 activation in cardiomyocytes expressing human TSC2(WT), TSC2(S1365A) or TSC2(S1365E) and stimulated with ET1 (10 nM) versus vehicle for 48 h. Data are mean ± s.d., n = 18 biologically independent samples, two-tailed unpaired Student’s t-test. b, From same experiment, PKG1 activation is found to be independent of the form of TSC2 expressed; n = 6 biologically independent samples, box and whisker and raw data plots. Data normalized to median for wild type (without ET1); P = 0.0004 for ET1 effect, P > 0.8 for TSC2 genotype effect by two-way ANOVA. c, Summary data for phosphorylated/total human TSC2(S1364) from the rat cardiomyocyte ET1-stimulation experiment shown in Fig. 1f. Data are mean ± s.d., n = 6 biologically independent samples from three experiments, one-way ANOVA with Tukey multiple comparisons test, #P = 0.003 versus TSC2(S1365E), P = 0.0003 versus TSC2(S1365A). d, Antibody raised against TSC2(S1365) (mouse) (equivalent to human TSC2(S1364)) shows increased TSC2 phosphorylation in rat cardiomyocytes transfected with human TSC2(WT), but not cells expressing human TSC2(S1365A) or human TSC2(S1365E). Summary from three independent replicates yielding n = 6 biologically independent samples, data are mean ± s.d., one-way ANOVA with Tukey multiple comparisons test, #P = 0.0002 versus TSC2(S1365E), P = 0.0007 versus TSC2(S1365A). The results are identical to those using mouse TSC2(S1365) (human TSC2(S1364)) mutants displayed in Fig. 1i, indicating that mutations at either serine (human sequence: S1364 or S1365; mouse sequence S1365 or 1366) prevents phosphorylation of the other and/or its detection by the phosphorylation-specific antibody. e, Direct TSC2 phosphorylation by recombinant PKG1α detected by autoradiography on human Flag–TSC2(WT) and Flag–TSC2(S1365A). Experiments were replicated three times (n = 6 biologically independent samples) with identical results. The result is identical to that in Fig. 1j with human HA–TSC2(S1364A). f, Direct TSC2 phosphorylation by PKG1α in lysates from TSC2 knockout HEK293 cells expressing human TSC2(WT) or human HA–TSC2(S1364A) or human Flag–TSC2(S1365A), PKG1α(M438G), and N6-benzyl-ATPγS, and probed for thiophosphate ester. Top, data with human TSC2(S1364) mutated; bottom, with human TSC2(S1365) mutated. The results are identical. n = 6 biologically independent samples for each assay.

  4. Extended Data Fig. 4 Human TSC2(S1364) or TSC2(S1365) mutated to glutamatic acid (S1364E or S1365E) suppresses ET1-stimulated cardiomyocyte hypertrophy and mTORC1 activation, whereas mutation to alanine (S1364A or S1365A) amplifies both.

    a, Nppb mRNA expression (pathological hypertrophy gene marker) in rat cardiomyocytes transfected with human Flag–TSC2(WT), Flag–TSC2(S1365A) or Flag–TSC2(S1365E) and then exposed to 48 h ET1 (to induce hypertrophy) or to vehicle. Activation of PKG1 by sildenafil reduces ET1-stimulated Nppb in TSC2(WT)-expressing cells, but not in cells expressing TSC2(S1365A) or TSC2(S1365E) mutants. TSC2(S1365E) expression depresses the increase in Nppb expression with ET1 stimulation, whereas TSC2(S1365A) expression enhances it. These results are nearly identical to those shown in Fig. 2a in which the human TSC2(S1364) (first serine of the duplet) was mutated. This shows that genetic modulation of either serine results in the same biological modulation of ET1 stimulation on growth and mTORC1 activity. Data are mean ± s.d., n = 6 biologically independent experiments, one-way ANOVA with Tukey multiple comparisons test, *P < 1 × 10−6 versus other TSC2(WT) groups; P = 0.002, ††P = 0.001  versus TSC2(S1365E), #P < 1 × 10−6 versus TSC2(S1365E) and TSC2(S1365A) and ET1, P < 1 × 10−6 versus TSC2(S1365E), §P < 1 × 10−6 versus TSC2(S1365E) and TSC2(WT) + ET1 and sildenafil. b, Summary analysis of immunoblots displayed in Fig. 1b. Values are normalized to TSC2(WT) treated with vehicle; data are mean ± s.d., n = 4 (LC3-II) or 6 (others) biologically independent experiments; one-way ANOVA with Tukey multiple comparisons test, *P ≤ 7 × 10−6 versus vehicle control, P < 1 × 10−6 versus TSC2(S1364A) and ET1, P < 5 × 10−6, #P = 0.01 versus TSC2(WT) and ET1. c, Example immunoblots from the same experiment as in a, showing changes in mTORC1 signalling proteins, p62 and LC3-II. ET1 stimulates phosphorylation of mTORC1 targets (p70S6K, 4E-BP1 and ULK1) and increases LC3-II and p62—consistent with mTORC1 activation and enhanced autophagy. Human TSC2(S1365E) reduces mTORC1 activation and p62 and increases LC3-II, whereas human TSC2(S1365A) does the opposite. This is identical to responses found using human S1364A and S1364E mutants (b and Fig. 2b), confirming the functional equivalency of either serine modification. Experiments were replicated 2–4 times, n = 4–8 biologically independent samples. d, Summary data for this experiment. Values normalized to TSC2(WT) treated with vehicle; data are mean ± s.d., n = 8 independent replicates for p70S6K and 4E-BP1, n = 6 for ULK1 and n = 4 for p62 and LC3-II. One-way ANOVA with Tukey multiple comparisons test, Results of pairwise comparisons: *P < 1 × 10−6 versus corresponding TSC2 genotype and vehicle, P ≤ 1 × 10−6 versus TSC2(WT) and ET1 and TSC2(S1365A) and ET1,#P = 0.003, P = 0.0001, P = 0.06, §P = 5 × 10−6, %P < 1 × 10−6 versus TSC2(WT) and ET1.

  5. Extended Data Fig. 5 Effects of TSC2(S1365A) and TSC2(S1365E) on mTORC1 activation in response to phenylephrine; PKG1 activation of autophagy requires in part its phosphorylation of TSC2; and amplification of mTORC1 stimulation in cells expressing S1365A TSC2 mutation requires RHEB.

    a, NRCMs expressing TSC2(WT), TSC2(S1365A) or TSC2(S1365E) (human TSC2(S1365) was modified) and exposed to vehicle or phenylephrine (PE, 100 mM) for 48 h. Left, example immunoblot for phospho-p70S6K and total protein. Right, summary data, normalized to TSC2(WT) without phenylephrine. Data are mean ± s.d., n = 6 biologically independent samples, Kruskal–Wallis Test with Dunn’s multiple comparisons test, *P = 0.003 versus corresponding vehicle; P = 0.0013 versus TSC2(S1365E) and ET1. b, Top, example immunoblots for LC3-II and p62 in lysates obtained from Tsc2 knockout MEFs transfected with either human TSC2(WT) or TSC2(S1364A) plasmids and then treated with ET1 (10 nM) with or without cGMP (50 μM). n = 3 biologically independent samples. Bottom, summary data, normalized to TSC2(WT) without cGMP and without ET1; data are mean ± s.d., one-way ANOVA with Tukey multiple comparisons test, *P = 0.01 versus TSC2(WT) without ET1 without cGMP, P = 0.02 versus TSC2(WT) and ET1 with cGMP, P < 1 × 10−6 versus TSC2(S1364A) without ET1 without cGMP and versus with ET1 with cGMP, #P = 2 × 10−6 versus TSC2(WT) with ET1, P = 5 × 10−5 versus TSC2(WT) and ET1 with cGMP, ††P = 1 × 10−6 versus TSC2(WT) without ET1 without cGMP and versus ET1 with cGMP, **P = 1 × 10−6 versus TSC2(WT) without ET1 without cGMP, §P = 0.002 versus TSC2(WT) with ET1, §§P < 1 × 10−6 versus TSC2(WT) with ET1 with sildenafil. c, Summary results for Fig. 2d. Rat cardiomyocytes transfected with RHEB or scrambled (Scr) siRNA, transfected with TSC2(WT), TSC2(S1365E) or TSC2(S1365A) plasmids (human TSC2(S1365) modified) and stimulated with ET1 for 48 h. Effect of gene silencing of Rheb on RHEB protein expression (right) and on phosphorylated/total p70S6K protein expression (left). n = 4 biologically independent experiments, data are mean ± s.d., values normalized to TSC2(WT) treated with vehicle and scramble siRNA. Each plot was analysed by one-way ANOVA with pairwise Tukey multiple comparisons test, P = 1 × 10−6 versus corresponding scramble siRNA without or with ET1, one-way ANOVA with Tukey multiple comparisons test, *P = 1 × 10−6 versus scramble siRNA and vehicle for TSC2(WT) and TSC2(S1365A), #P = 0.00002 versus scramble siRNA and ET1 for TSC2(WT) or TSC2(S1365A).

  6. Extended Data Fig. 6 Tsc2S1365A/S1365A knock-in mouse genotyping; expression of TSC2 with or without pressure overload; and effect on pressure-overload-stimulated hypertrophy, autophagy and mTORC1 activation.

    a, Mouse Tsc2S1365A/S1365A knock-in genotyping by PCR detects a unique sequence based on the mutated residue as a 206-base pair (BP) fragment. This signature was used for genotyping. b, Immunoblot of TSC2 protein from Tsc2S1365A/S1365A and Tsc2WT/WT (littermate controls) for sham and pressure-overload-treated groups. There is no difference in expression levels among these groups or conditions. Experiments were repeated independently three times with identical results. c, Nppa mRNA expression normalized to Gapdh mRNA expression in Tsc2WT/WT versus Tsc2S1365A/S1365A myocardium before and after chronic pressure overload. n = 8 biologically independent experiments, data are mean ± s.d., one-way ANOVA with Tukey multiple comparisons test, *P = 2 × 10−5 versus sham, P < 1 × 10−6 versus sham, P = 3 × 10−6 versus Tsc2WT/WT pressure overload (PO). d, Immunoblots of LC3-II from Tsc2WT/WT, Tsc2S1365A/WT and Tsc2S1365A/S1365A myocardium from mice subjected to sham or pressure overload and treated with vehicle or sildenafil. Pressure-overload-stimulated LC3-II expression is lacking in Tsc2S1365A/WT and Tsc2S1365A/S1365A, but is recovered in Tsc2S1365A/WT mice after sildenafil treatment. It increases further in Tsc2WT/WT mice after pressure overload with sildenafil treatment. Experiments were replicated twice providing 4 biologically independent samples, data are mean ± s.d.; data normalized to the mean of Tsc2WT/WT sham, one-way ANOVA with Tukey test for multiple comparisons, *P < 1 × 10−6 versus respective sham and versus pressure overload in other two groups, P < 1 × 10−6 versus respective sham and versus Tsc2S1365A/S1365A pressure overload and sildenafil, P = 0.0013 versus Tsc2S1365A/WT pressure overload and sildenafil, P < 1 × 10−6 versus sham and Tsc2S1365A/S1365A pressure overload and sildenafil. e, Summary data for immunoblots displayed in Fig. 3e, f. n = 4 biologically independent experiments, data are mean ± s.e.m., data normalized to mean of Tsc2WT/WT sham; one-way ANOVA with Tukey multiple comparisons test, *P < 1 × 10−6 versus respective sham and pressure overload with sildenafil treatment; P = 0.0002 versus sham, P < 1 × 10−6 versus respective sham and Tsc2WT/WT and pressure overload, #P < 1 × 10−6 versus Tsc2WT/WT and Tsc2S1365A/WT and pressure overload with sildenafil treatment.

  7. Extended Data Fig. 7 Tsc2S1365A/S1365A knock-in mice display significantly increased mTORC1 but no change in mTORC2 activation; depressed autophagy in Tsc2S1365A/S1365A mice subjected to pressure overload is reversed by mTOR inhibition with everolimus.

    a, Immunoblots and summary quantification for mTORC2 targets from Tsc2WT/WT and Tsc2S1365A/S1365A mice subjected to sham or pressure-overload surgeries and treated with sildenafil or vehicle. n = 4 biologically independent experiments, box and whisker plots (median) with individual data are shown; data normalized to median of Tsc2WT/WT sham. One-way ANOVA with Tukey multiple comparisons test. P70S6K is shown at the top as an mTORC1 control, showing increased phosphorylation with pressure overload that is greater and unresponsive to sildenafil in Tsc2S1365A/S1365A mice. *P = 0.002 versus sham, P = 0.04 versus pressure overload, P = 0.03 versus sham. However, there were no significant changes (P ≥ 0.62 between conditions within genotype) in the expression of mTORC2 substrates: phosphorylated (S473)/total AKT, phosphorylated (T24/T32)/total FOXO1/3 and phosphorylated(T346)/total NRDG1. b, LC3-II expression is unaltered while p62 expression increases from pressure overload in Tsc2S1365A/S1365A myocardium, indicating suppression of autophagy. Both are reversed by treatment with the mTORC1 inhibitor everolimus. n = 6 biologically independent animal experiments, data are mean ± s.d., one-way ANOVA with Tukey multiple comparisons test, *P ≤ 3 × 10−5 versus other two groups. Data are normalized to the mean of sham control.

  8. Extended Data Fig. 8 Generation of Tsc2S1365E/S1365E knock-in mice and impact on pressure-overload-induced mTORC1 activation, autophagy and autophagic flux in vivo.

    a, Strategy and guide RNA for CRISPR–Cas9 protocol to generate Tsc2S1365E/S1365E knock-in mice. b, Summary data for Fig. 4f. Phosphorylated/total p70S6K (n = 6 biologically independent experiments), p62/α-tubulin and LC3-II/total protein (n = 4 biologically independent experiments, data are mean ± s.d., values are normalized to Tsc2WT/WT sham), two-way ANOVA with Sidak’s multiple comparisons test, *P < 1 × 10−6 versus Tsc2WT/WT sham, P = 0.0012 versus Tsc2S1365E/WT with pressure overload, and P = 0.0001 versus Tsc2S1365E/S1365E with pressure overload, P = 0.0008 versus Tsc2S1365E/WT sham, P = 0.001 versus Tsc2S1365E/S1365E sham; for p62, #P ≤ 1 × 10−6 versus all other groups, for LC3-II, §P = 0.018 and P = 0.0003 versus pressure overload Tsc2S1365E/WT and pressure overload Tsc2S1365E/S1365E, respectively, **P = 0.02 versus pressure overload Tsc2S1365E/S1365E and P = 0.0002 versus sham, ##P < 1 × 10−6 versus sham. c, Example immunoblot and summary results for Tsc2WT/WT, Tsc2S1365E/S1365E and Tsc2S1365A/S1365A mice treated with BFA or vehicle. Myocardium was assayed for LC3-II, with higher expression in the presence of BFA, indicating greater autophagic flux. Summary data, values normalized to Tsc2WT/WT vehicle, data are mean ± s.d., n = 4 biologically independent experiments; two-way ANOVA: P < 1 × 10−6 for BFA effect, P = 0.003 for Tsc2 genotype effect, and P = 0.002 for interaction; Sidak’s pairwise multiple comparisons test: *P = 0.0008 versus Tsc2WT/WT vehicle, P < 1 × 10−6 versus Tsc2S1365E/S1365E vehicle and P = 0.008 versus Tsc2S1365A/S1365A + BFA, P = 0.05 versus Tsc2WT/WT + BFA.

  9. Extended Data Table 1 Cardiac morphometry and function analyses

Supplementary information

  1. Supplementary Figures

    This file contains the raw gels for every immunoblot shown in the paper and the Extended Data Figures

  2. Reporting Summary

  3. Supplementary Table

    This file contains a list of all the commercial antibodies used in the study, their catalogue number and information provided by the manufacturer as to how each was validated

Source data

About this article

Publication history




Issue Date



Further reading


By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.