Mammalian Target of Rapamycin Signaling in Cardiac Physiology and Disease Sebastiano Sciarretta, Massimo Volpe and Junichi Sadoshima Circ Res. 2014;114:549-564 doi: 10.1161/CIRCRESAHA.114.302022 Circulation Research is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231 Copyright © 2014 American Heart Association, Inc. All rights reserved. Print ISSN: 0009-7330. Online ISSN: 1524-4571
The online version of this article, along with updated information and services, is located on the World Wide Web at: http://circres.ahajournals.org/content/114/3/549
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Review Mammalian Target of Rapamycin Signaling in Cardiac Physiology and Disease Sebastiano Sciarretta, Massimo Volpe, Junichi Sadoshima
Abstract: The protein kinase mammalian or mechanistic target of rapamycin (mTOR) is an atypical serine/threonine kinase that exerts its main cellular functions by interacting with specific adaptor proteins to form 2 different multiprotein complexes, mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2). mTORC1 regulates protein synthesis, cell growth and proliferation, autophagy, cell metabolism, and stress responses, whereas mTORC2 seems to regulate cell survival and polarity. The mTOR pathway plays a key regulatory function in cardiovascular physiology and pathology. However, the majority of information available about mTOR function in the cardiovascular system is related to the role of mTORC1 in the unstressed and stressed heart. mTORC1 is required for embryonic cardiovascular development and for postnatal maintenance of cardiac structure and function. In addition, mTORC1 is necessary for cardiac adaptation to pressure overload and development of compensatory hypertrophy. However, partial and selective pharmacological and genetic inhibition of mTORC1 was shown to extend life span in mammals, reduce pathological hypertrophy and heart failure caused by increased load or genetic cardiomyopathies, reduce myocardial damage after acute and chronic myocardial infarction, and reduce cardiac derangements caused by metabolic disorders. The optimal therapeutic strategy to target mTORC1 and increase cardioprotection is under intense investigation. This article reviews the information available regarding the effects exerted by mTOR signaling in cardiovascular physiology and pathological states. (Circ Res. 2014;114:549-564.) Key Words: autophagy
■
heart
■
hypertrophy ■ ischemia ■ metabolism complex 1 ■ sirolimus
T
he protein kinase mammalian target of rapamycin (mTOR) was purified and characterized for the first time in mammalian cells in the independent works conducted by Brown et al,1 Sabatini et al,2 and Sabers et al3 in 1994 and 1995. mTOR is an atypical serine/threonine kinase that exerts its main cellular functions by interacting with specific adaptor proteins to form 2 distinct multiprotein complexes, mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2).4–8 mTOR signaling plays a key role in the regulation of cell homeostasis and stress responses. mTORC1 is a master regulator of protein synthesis, cell growth and proliferation, ribosomal and mitochondrial biogenesis, autophagy, and metabolism. In addition, mTORC1 inhibition during stress is an adaptive response that promotes the upregulation of stress responsive mechanisms. Whereas, mTORC2 seems to regulate cell survival and polarity4–8 (Figure 1). Studies of animal models with loss of function of the components of mTOR complexes have indicated that mTOR is involved in the regulation of embryonic cardiovascular development and in the control of vital cellular processes necessary for normal postnatal growth and maintenance of cardiac function. Cardiac deletion of mTOR is associated with a high
■
mechanistic target of rapamycin
rate of embryonic lethality, and cardiac disruption of the components of mTORC1 is associated with cardiac dilation, dysfunction, apoptosis, mitochondrial and metabolic derangements, heart failure, and, ultimately, mortality in the postnatal stage.9–12 In addition, complete genetic disruption of mTORC1 impairs the ability of the heart to respond to pressure overload and to undergo compensatory hypertrophy, resulting in the development of dilated cardiomyopathy.10 However, the available evidence suggests that partial and selective inhibition of mTORC1 is cardioprotective during aging and cardiac stress. mTORC1 inhibition extends the life span of mice.13–15 It also reduces cardiac hypertrophy and improves cardiac function in the presence of pressure overload16–19 and genetic cardiomyopathies20–22 and reduces ischemic injury after acute23,24 and chronic myocardial infarction.19,25,26 Finally, the inhibition of mTORC1 reactivates cardiac autophagy, which is impaired in the presence of obesity and metabolic syndrome.23 These results are likely dependent on the fact that partial inhibition of mTORC1 eliminates the detrimental effects of the maladaptive functions of mTORC1 during cardiac stress, while maintaining its physiological functions. In this regard, it is known that rapalogs do not inhibit all the functions of mTORC1.27
Original received October 23, 2013; revision received November 28, 2013; accepted December 2, 2013. In November 2013, the average time from submission to first decision for all original research papers submitted to Circulation Research was 14.6 days. From the Cardiovascular Research Institute, Department of Cell Biology and Molecular Medicine, Rutgers New Jersey Medical School, Newark, NJ (S.S., J.S.); IRCCS Neuromed, Pozzilli, Italy (S.S., M.V.); and Division of Cardiology, Department of Clinical and Molecular Medicine, Faculty of Medicine and Psychology, University Sapienza, Rome, Italy (M.V.). Correspondence to Junichi Sadoshima, MD, PhD, Department of Cell Biology and Molecular Medicine, Rutgers New Jersey Medical School, 185 South Orange Ave, Medical Science Building G-609, Newark, NJ 07103. E-mail [email protected] © 2014 American Heart Association, Inc. Circulation Research is available at http://circres.ahajournals.org
DOI: 10.1161/CIRCRESAHA.114.302022
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550 Circulation Research January 31, 2014
Nonstandard Abbreviations and Acronyms 4E-BP1 Akt AMPK Atg eIF4E ERK GSK NF-κB PGC-1α PI3K PRAS40 Raptor Rheb Rictor S6K SGK-1 TAC TSC ULK
eIF4E-binding protein-1 protein kinase B adenosine monophosphate-activated protein kinase autophagy-related gene eukaryotic translation initiation factor 4E extracellular signal regulated kinase glycogen synthase kinase nuclear factor-κB peroxisome proliferator-activated receptor γ coactivator-1α phosphoinositide 3 kinase proline-rich Akt substrate 40 regulatory-associated protein of mammalian target of rapamycin (mTOR) Ras homolog enriched in brain rapamycin-insensitive companion of mTOR S6 kinase serum- and glucocorticoid-induced protein kinase-1 transverse aortic constriction tuberous sclerosis protein unc-51-like kinase
The degree of mTORC1 inhibition and the mTORC1 physiological functions needed to be preserved to convert mTORC1 inhibition from detrimental into beneficial during cardiac stress are unclear. In contrast, information about the pathophysiological functions of mTORC2 in the heart is still scarce. This article reviews and interprets the evidence currently available regarding the role of mTOR signaling pathway in cardiovascular physiology and disease (Table).
Biology of the mTOR Pathway The mTOR kinase is an atypical serine/threonine kinase of 289 kDa and belongs to the family of phosphoinositide
Figure 1. General cellular functions of mammalian target of rapamycin complex 1 (mTORC1) and mTORC2. The figure summarizes the most well-characterized functions of mTORC1 and mTORC2. mTORC1 regulates protein synthesis and autophagy in response to growth factors and stress. mTORC2 is known to regulate cell growth, survival, and polarity. Akt indicates protein kinase B; and AMPK, adenosine monophosphateactivated protein kinase.
3-kinase. It is encoded by a single gene in mammals, but it exerts its main cellular functions by forming mTORC1 and mTORC2 through assembly with specific adaptor proteins.4–8 mTORC1 and mTORC2 signaling pathways are evolutionarily conserved, and these represent the functional homologs of yeast TORC1 and TORC2. However, TOR1 and TOR2 are encoded by distinct genes in yeast, as first identified in Michael Hall’s laboratory in 1991.28 mTOR was isolated and cloned as a physical target of rapamycin through a cellular screening aimed at identifying the binding proteins of FK506-binding protein of 12 kDa–rapamycin complex.1–3 Rapamycin and its analogs bind to the cytosolic FK506-binding protein of 12 kDa, thereby forming a protein complex that only targets a specific domain of the mTOR protein when it is part of mTORC1. As a consequence of rapamycin–FK506-binding protein of 12 kDa binding, mTORC1 activity is strongly inhibited.4,6 Conversely, mTORC2 is relatively insensitive to rapamycin, although it has been demonstrated that prolonged treatment with rapamycin can also reduce the activity of mTORC2 by disrupting the complex.29 To date, proteins that are known to bind to mTOR in mTORC1 include regulatory-associated protein of mTOR (Raptor),30 mammalian lethal with sec-13 protein 8,31 proline-rich protein kinase B (Akt) substrate 40 (PRAS40),32 DEP domain-containing mTOR-interacting protein,33 and Tel two interacting protein 1/Tel2.34 Whereas, mTORC2 contains mTOR, the scaffold protein rapamycin-insensitive companion of mTOR (Rictor),35 stress-activated map kinase-interacting protein 1,36 protein observed with rictor,37 mammalian lethal with sec-13 protein 8,31 DEP domain-containing mTOR- interacting protein,33 and Tel two interacting protein 1/Tel2.34 The main functions of proteins forming the mTOR complexes are to maintain the integrity of the complexes, regulate their complex subcellular localization, and control their complex interactions with substrates and regulators4–8 (Figure 2). Evidence regarding the biological functions of mTORC1 and mTORC2 suggests that they have distinct cellular functions, substrates, and regulators.4–8 However, based on available evidence, it is difficult to distinguish the function of 1 complex completely from the other. It has been shown that the 2 complexes are functionally interconnected, and most of the studies that investigated the role of mTORC1 in the regulation of cellular homeostasis did not precisely rule out the involvement of mTORC2 in these mechanisms. In general, much more is known about the biology of mTORC1 than that of mTORC2 (Figure 2). mTORC1 plays a crucial role in the regulation of cellular homeostasis, growth, and response to stress. The main functions of mTORC1 are promotion of protein synthesis and, subsequently, cellular growth. The most studied substrates of mTORC1 are S6 kinase-1 (S6K1) and eukaryotic translation initiation factor 4E (eIF4E)-binding protein-1 (4E-BP1). mTORC1 phosphorylates and activates S6K1, which in turn promotes mRNA biogenesis and activates the protein translation process. In contrast, mTORC1 inhibits 4E-BP1 and allows the formation of the eIF4F complex that triggers cap-dependent translation.4–8 Additionally, mTORC1 promotes protein synthesis through the translation of 5′ terminal oligopyrimidine tract mRNAs and the promotion of ribosomal biogenesis.4–8 This mechanism explains why mTORC1 activity is often found to be increased in cancer cells.4–8
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Sciarretta et al mTOR Signaling in Cardiovascular System 551 Table. Main Studies That Investigated the Role of mTOR Kinase in the Regulation of Cardiac Physiology and Response to Stress In Vivo Cardiac Condition Development/ physiological conditions
Aging
Cardiac hypertrophy
Cardiac ischemia and reperfusion
Study
Type of mTOR Modulation and Animal Model
Effect on the Heart
Constitutive α-MHC-CRE–mediated mTOR gene deletion
High embryonic lethality due to cardiac failure, massive cardiac dilation, dysfunction, heart failure, early mortality in the postnatal stage, and metabolic derangements
Zhang et al10
Tamoxifen-induced α-MHC-CRE–mediated mTOR deletion during adulthood
Massive cardiac dilation, apoptosis, autophagy, mitochondrial abnormalities, sarcomere disarray, cardiac dysfunction, heart failure, and early mortality
Shende et al11
Tamoxifen-induced α-MHC-CRE–mediated mTOR deletion during adulthood
Massive cardiac dilation, apoptosis, autophagy, mitochondrial abnormalities, sarcomere disarray, metabolic abnormalities, cardiac dysfunction, heart failure, and early mortality
Tamai et al12
Constitutive α-MHC-CRE–mediated rheb1 gene deletion
Reduction of mTORC1 activity 5 days after birth, defect in physiological cardiac growth, cardiac dilation, dysfunction, heart failure, mortality ≤10 days after birth
Flynn et al14
Pharmacological mTOR inhibition using rapamycin
Reduced age-induced cardiac inflammation and fibrosis, upregulation of energy metabolism
Zhou et al80
Systemic GSK-3α deletion
Cardiac hypertrophy, dysfunction, and sarcomere abnormalities during aging through mTOR activation and autophagy inhibition
Zhang et al10
Tamoxifen-induced α-MHC-CRE–mediated mTOR deletion during adulthood and pressure overload
Inhibition of compensatory hypertrophy, inhibition of protein synthesis, rapid cardiac dilation, apoptosis, cardiac dysfunction, and heart failure
Shende et al11
Tamoxifen-induced α-MHC-CRE–mediated mTOR deletion during adulthood and pressure overload
Inhibition of compensatory hypertrophy, inhibition of protein synthesis, rapid cardiac dilation, cardiac dysfunction, and heart failure
Shioi et al16
mTORC1 inhibition using rapamycin and then pressure overload
Inhibition of pathological cardiac hypertrophy and preservation of cardiac function
McMullen et al17
mTORC1 inhibition using rapamycin in mice with established TAC-induced cardiac hypertrophy
Regression of compensated and decompensated hypertrophy, improved cardiac function in the latter
Völkers et al18
AAV9-mediated PRAS40 overexpression in hearts subjected to pressure overload
Prevention of TAC-induced pathological hypertrophy and myocardial fibrosis, maintenance of cardiac function
Wu et al19
Constitutive α-MHC-CRE–mediated heterozygous rheb1 gene deletion and then pressure overload
Reduction of TAC-induced hypertrophy and fibrosis
Marin et al20
Mouse model of LEOPARD disease treated with rapamycin
Regression of hypertrophic cardiomyopathy and cardiac disarray
Choi et al22; Ramos et al21
Mouse model of cardiomyopathy caused by Lamin A/C mutation treated with pharmacological mTORC1 inhibition
Autophagy reactivation and improved cardiac function, muscle dystrophy, and increased survival
Song et al99
α-MHC-CRE–mediated mTOR overexpression
No increase in cardiac mass, preservation of cardiac function and reduced myocardial inflammation through inhibition of NF-κB signaling
Sciarretta et al23
Pharmacological inhibition of mTORC1 during prolonged ischemia, prolonged ischemia in mice with α-MHC-CRE–mediated Rheb overexpression
Inhibition of ischemia-induced mTORC1 downregulation and autophagy activation, increased infarct size after ischemia that is reversed by rapamycin treatment
Zhai et al24
Mice with α-MHC-CRE–mediated dominant negative GSK-3β overexpression and systemic heterozygous GSK-3β deletion subjected to prolonged ischemia and I/R
GSK-3β is activated during prolonged ischemia but inhibited during reperfusion; genetic inhibition of GSK-3β increases ischemic injury after prolonged ischemia but reduces reperfusion injury through a deregulated activation of mTORC1
Buss et al25
mTORC1 inhibition with everolimus during chronic myocardial infarction
Reduction of infarct size, pathological growth, and cardiac dilation; improvement of cardiac function; activation of autophagy
AAV9-mediated PRAS40 cardiac overexpression and Rictor knockdown in mice subjected to chronic myocardial infarction
Overexpression of PRAS40 inhibits mTORC1, reduces ischemic injury, apoptosis and cardiac remodeling, and improves cardiac function in an mTORC2-dependent manner; Rictor knockdown causes deterioration of cardiac function and remodeling after myocardial infarction
Di et al111
Rapamycin and S6K inhibitors during chronic myocardial infarction
Reduction of cardiac ischemic remodeling and cardiomyocte apoptosis
Aoyagi et al119
α-MHC-CRE–mediated mTOR overexpression and I/R
Inhibition of cardiac remodeling after I/R in vivo, and reduction of necrosis and myocardial inflammation after I/R ex vivo (Continued )
Zhu et al
9
Völkers et al26
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552 Circulation Research January 31, 2014 Table. Continued Cardiac Condition Metabolic disorders
Study Wang et al
Type of mTOR Modulation and Animal Model
Effect on the Heart
Rapamycin treatment during high-fat diet– induced obesity
Akt and mTOR are activated in the vasculature of obese animals; rapamycin inhibits endothelial senescence and the susceptibility to peripheral ischemia in obese animals
Sciarretta et al23
Rapamycin treatment and partial inducible mTOR genetic disruption in mice with high-fat diet–induced obesity and metabolic syndrome subjected to prolonged ischemia
Ischemia-induced autophagy activation is inhibited in the hearts of obese animals through deregulated activation of Rheb and mTORC1; infarct size after ischemia is larger in obese animals; pharmacological and partial genetic inhibition of mTOR rescue autophagy and reduce ischemic injury in obese mice
Li et al126
Swine model of metabolic syndrome
Increased cardiac mTOR level and reduction of cardiac autophagy that is proportional to the degree of derangements of cardiac structure and function
Guo et al127
Rapamycin in mice with high-fat diet–induced obesity
Increased mTOR activity and reduced autophagosome formation in the hearts of obese mice, associated with a reduction of cardiac function; cardiac function is rescued by rapamycin and deteriorated by genetic adiponectin disruption
Xu et al128
Mice with systemic Akt2 knockout subjected to high-fat diet
mTOR activation and disruption of autophagic flux in the hearts of obese mice; Akt2 deletion rescues autophagic flux and cardiac dysfunction in obese animals
Xu et al129
Streptozotocin-induced diabetes mellitus
Cardiac autophagosome formation and flux are impaired in diabetic mice; these effects are associated with increased mTOR activity
124
AAV9 indicates adeno-associated virus 9; Akt, protein kinase B; GSK-3β, glycogen synthase kinase-3β; I/R, ischemia/reperfusion; LEOPARD, Lentigines, ECG conduction abnormalities, Ocular hypertelorism, Pulmonic stenosis, Abnormal genitalia, Retardation of growth and sensorineural Deafness; mTOR, mammalian target of rapamycin; mTORC1, mTOR complex 1; NF-κB, nuclear factor-κB; PRAS40, proline-rich Akt substrate 40; Rheb, Ras homolog enriched in brain; Rictor, rapamycininsensitive companion of mTOR; S6K, S6 kinase; and TAC, transverse aortic constriction.
mTORC1 also promotes cell proliferation by promoting the de novo synthesis of cellular membrane lipids through the sterol regulatory element-binding protein 1/2–dependent expression of lipogenic genes.7,38 However, mTORC1 not only activates anabolic processes but also inhibits catabolic processes. mTORC1 strongly inhibits autophagy, an evolutionarily conserved intracellular bulk degradation process responsible for cellular degradation of proteins and organelles.6,7,39,40 mTORC1 seems to regulate autophagy both at post-translational and transcriptional levels. It phosphorylates the autophagic protein unc-51-like kinase (ULK) 1/2, thereby inhibiting the macrocomplex ULK1/autophagy-related gene (Atg) 13/focal adhesion kinase family interacting protein-200 that promotes autophagosome formation.41 Additionally, mTORC1 activation inhibits the expression of autophagic proteins, particularly Atg7,23 which is crucial for the initiation of the autophagic process.39 It has been shown that mTOR significantly inhibits the p73 factor and the transcription factor EB that can induce autophagy through the upregulation of autophagic proteins such as Atg7.42–44 mTORC1 also plays an important role in the regulation of cellular metabolism. mTORC1 promotes mitochondrial biogenesis and oxidative metabolism through the peroxisome proliferator–activated receptor γ coactivator-1α (PGC-1α)-mediated activation of the transcription factor Ying-Yang 1.45 mTORC1 can activate glycolysis and the oxidative pentose phosphate pathway through the activation of hypoxia-inducible factor-1α and sterol regulatory element–binding protein 1/2, respectively.46 Finally, mTORC1 activity is inhibited by acid deprivation, energy stress, hypoxia, ER stress, and genotoxic stress.4–8 Under these conditions, mTORC1 inhibition allows the upregulation
of stress responsive mechanisms that limit cell death, such as reduction of protein synthesis, autophagy, cell cycle arrest, and DNA repair. The Akt and adenosine monophosphate–activated protein kinase (AMPK) pathways represent the most well- characterized regulators of mTORC1. In the presence of nutrients and growth factors such as insulin, insulin growth factor-1, platelet-derived growth factor, and epidermal growth factor, Akt is activated and, in turn, phosphorylates and activates mTOR or inhibits PRAS40, an endogenous mTORC1 inhibitor.4–8,32,47 Whereas, AMPK, which is inactive in the presence of nutrients and high ATP levels, is strongly activated during energy deprivation and other types of cellular stress. AMPK activates the tuberous sclerosis protein (TSC) 1/TSC2 complex,48 which displays a strong GTPase activity and inhibits the small GTP-binding protein Ras homolog enriched in brain (Rheb), a direct mTORC1 activator.49,50 Glycogen synthase kinase (GSK) 3β can also activate the TSC1/ TSC2 complex and inhibit mTORC1 during energy stress.48 Alternatively, regulated in development and DNA damage response 1, which is upregulated during hypoxia, activates TSC2 independently of AMPK and GSK-3β.51 Rheb can also be inhibited by p38-regulated/activated kinase during energy deprivation, independently of TSC2.52 Conversely, the Akt, extracellular signal regulated kinase (ERK) 1/2, and inhibitor of NF-κB kinase-β pathways inhibit the TSC1/TSC2 complex in response to growth factors and cytokines.53–55 Recently, a new mechanism promoting the activation of mTORC1 has been elucidated. In the presence of amino acids, Rag GTPases are activated and mediate mTORC1 translocation to lysosome membranes, where mTORC1 is activated by Rheb.56,57
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Sciarretta et al mTOR Signaling in Cardiovascular System 553
Figure 2. General overview of the mammalian target of rapamycin (mTOR) signaling pathway. The dashed line signifies that rapamycin inhibits mTOR complex 2 (mTORC2) in specific cell types or after prolonged treatment. 4EB-P1 indicates eukaryotic translation initiation factor 4E (eIF4E)-binding protein-1; Akt, protein kinase B; AMPK, adenosine monophosphate-activated protein kinase; Atg7, autophagy-related gene 7; DEPTOR, DEP domain-containing mTOR-interacting protein; ERK1/2, extracellular signal regulated kinase 1/2; FKBP12, FK506-binding protein of 12 kDa; GSK-3β, glycogen synthase kinase-3β; HIF-1, hypoxia-inducible factor-1α; IKKβ, inhibitor of NF-κB kinase-β; mLST8, mammalian lethal with sec-13 protein 8; PGC-1α, peroxisome proliferator-activated receptor γ coactivator-1α; PI3K, phosphoinositide 3 kinase; PKC-α, protein kinase C-α; PRAK, p38-regulated/activated kinase; PRAS40, proline-rich Akt substrate 40; PROTOR, protein observed with rictor; Raptor, regulatory-associated protein of mTOR; REDD1, regulated in development and DNA damage response 1; Rheb, Ras homolog enriched in brain; RhoA, Ras homolog gene family, member A; S6K1, S6 kinase-1; SGK1, serum- and glucocorticoid-induced protein kinase-1; SREBP1/2, sterol regulatory element-binding protein 1/2; TFEB, transcription factor EB; TSC1/2, tuberous sclerosis protein 1/2; Tt1, Tel two interacting protein 1; ULK1/2, unc-51-like kinase 1/2; and YY1, Ying-Yang 1.
This process seems to be negatively regulated by microtubuleassociated protein/microtubule affinity-regulating kinase 4, independently of Rheb activity.58 mTORC2 can be activated by insulin and growth factors, whereas it is relatively insensitive to nutrient deprivation.5,6 The phosphoinositide 3 kinase (PI3K) pathway was shown to activate mTORC2,59 and it seems that the TSC1/2 complex does as well.60 mTORC1 seems to inhibit mTORC2 through phosphorylation of Rictor,61 suggesting that mTORC1 and mTORC2 are functionally interconnected. This hypothesis is also strongly supported by the fact that the best known substrate of mTORC2 is Akt, which is phosphorylated at serine 473, particularly in response to insulin.62,63 The biological importance of mTORC2-dependent phosphorylation of Akt is not yet understood, because it has been shown that mTORC2 is dispensable for Akt phosphorylation in certain cell types and conditions.64 Both protein kinase C-α65 and serum- and glucocorticoid-induced protein kinase-1 (SGK-1)66 have been shown to be substrates of mTORC2, despite mTORC1 also being shown to regulate SGK-1.67 Whether SGK-1 exerts different functions in cardiomyocytes when it is activated by mTORC1 or mTORC2 is unclear. The most studied function
of mTORC2 is the regulation of survival and growth, likely through the regulation of Akt and SGK-1. SGK-1 has been shown to promote cardiomyocyte survival while inhibiting hypertrophy,68 whereas SGK-1 chronic activation during heart failure is detrimental.69 mTORC2 also regulates cell polarity and cytoskeletal organization through the regulation of protein kinase C-α and Ras homolog gene family member A.65 Protein kinase C-α has also been shown to negatively regulate cardiac contractility.70
Role of mTOR Signaling in the Regulation of Cardiac Homeostasis and Physiological Growth Given its myriad cellular functions, it is not surprising that mTOR kinase is necessary for normal regulation of cardiomyocyte homeostasis and growth during both development and postnatal period (Figure 3). Systemic mTOR- and Raptor- knockout embryos die early during development, right after implantation.63 However, embryos with constitutive α-MHCCre–mediated mTOR deletion also display a dramatic mortality starting around E13.5, with only 8% of embryos surviving the developmental stage. Cardiac-specific mTOR-knockout embryos present a significant reduction in cardiomyocyte
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554 Circulation Research January 31, 2014 proliferation and an increase in apoptosis, with a 34% reduction in cardiomyocyte number. As a result, cardiac-specific mTOR-knockout embryos present cardiac dilation and dysfunction, with signs of terminal cardiac failure.9 Consistent with these results, systemic deletion of the rheb1 gene that extensively reduces mTORC1 activity is embryonically lethal, most likely because of cardiac defects such as ventricular wall thinning and cardiomyocyte apoptosis.71 Global rictor gene deletion that selectively disrupts mTORC2 is also lethal in the developmental stage, and embryos with rictor deletion display significant cardiovascular abnormalities.35 Thus, both mTORC1 and mTORC2 are highly important for cardiac development and embryo survival. The mTOR pathway also seems vital for the maintenance of cardiac structure and function in the postnatal period and adulthood. Mice with α-MHC-Cre–mediated cardiac mTOR disruption that are born alive die within a few weeks after birth from massive cardiac dilation, dysfunction, and heart failure.9 mTOR-knockout mice present with derangements in fatty acid metabolism in the heart. Inducible cardiac-specific mTOR deletion in adulthood also leads to cardiac dysfunction and heart failure, with chamber dilation and wall thinning. mTOR-knockout mice display massive apoptosis, fibrosis, autophagy, mitochondrial abnormalities and dysfunction, sarcomere disarray, and ultimately death
The online version of this article, along with updated information and services, is located on the World Wide Web at: http://circres.ahajournals.org/content/114/3/549
Permissions: Requests for permissions to reproduce figures, tables, or portions of articles originally published in Circulation Research can be obtained via RightsLink, a service of the Copyright Clearance Center, not the Editorial Office. Once the online version of the published article for which permission is being requested is located, click Request Permissions in the middle column of the Web page under Services. Further information about this process is available in the Permissions and Rights Question and Answer document. Reprints: Information about reprints can be found online at: http://www.lww.com/reprints Subscriptions: Information about subscribing to Circulation Research is online at: http://circres.ahajournals.org//subscriptions/
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Review Mammalian Target of Rapamycin Signaling in Cardiac Physiology and Disease Sebastiano Sciarretta, Massimo Volpe, Junichi Sadoshima
Abstract: The protein kinase mammalian or mechanistic target of rapamycin (mTOR) is an atypical serine/threonine kinase that exerts its main cellular functions by interacting with specific adaptor proteins to form 2 different multiprotein complexes, mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2). mTORC1 regulates protein synthesis, cell growth and proliferation, autophagy, cell metabolism, and stress responses, whereas mTORC2 seems to regulate cell survival and polarity. The mTOR pathway plays a key regulatory function in cardiovascular physiology and pathology. However, the majority of information available about mTOR function in the cardiovascular system is related to the role of mTORC1 in the unstressed and stressed heart. mTORC1 is required for embryonic cardiovascular development and for postnatal maintenance of cardiac structure and function. In addition, mTORC1 is necessary for cardiac adaptation to pressure overload and development of compensatory hypertrophy. However, partial and selective pharmacological and genetic inhibition of mTORC1 was shown to extend life span in mammals, reduce pathological hypertrophy and heart failure caused by increased load or genetic cardiomyopathies, reduce myocardial damage after acute and chronic myocardial infarction, and reduce cardiac derangements caused by metabolic disorders. The optimal therapeutic strategy to target mTORC1 and increase cardioprotection is under intense investigation. This article reviews the information available regarding the effects exerted by mTOR signaling in cardiovascular physiology and pathological states. (Circ Res. 2014;114:549-564.) Key Words: autophagy
■
heart
■
hypertrophy ■ ischemia ■ metabolism complex 1 ■ sirolimus
T
he protein kinase mammalian target of rapamycin (mTOR) was purified and characterized for the first time in mammalian cells in the independent works conducted by Brown et al,1 Sabatini et al,2 and Sabers et al3 in 1994 and 1995. mTOR is an atypical serine/threonine kinase that exerts its main cellular functions by interacting with specific adaptor proteins to form 2 distinct multiprotein complexes, mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2).4–8 mTOR signaling plays a key role in the regulation of cell homeostasis and stress responses. mTORC1 is a master regulator of protein synthesis, cell growth and proliferation, ribosomal and mitochondrial biogenesis, autophagy, and metabolism. In addition, mTORC1 inhibition during stress is an adaptive response that promotes the upregulation of stress responsive mechanisms. Whereas, mTORC2 seems to regulate cell survival and polarity4–8 (Figure 1). Studies of animal models with loss of function of the components of mTOR complexes have indicated that mTOR is involved in the regulation of embryonic cardiovascular development and in the control of vital cellular processes necessary for normal postnatal growth and maintenance of cardiac function. Cardiac deletion of mTOR is associated with a high
■
mechanistic target of rapamycin
rate of embryonic lethality, and cardiac disruption of the components of mTORC1 is associated with cardiac dilation, dysfunction, apoptosis, mitochondrial and metabolic derangements, heart failure, and, ultimately, mortality in the postnatal stage.9–12 In addition, complete genetic disruption of mTORC1 impairs the ability of the heart to respond to pressure overload and to undergo compensatory hypertrophy, resulting in the development of dilated cardiomyopathy.10 However, the available evidence suggests that partial and selective inhibition of mTORC1 is cardioprotective during aging and cardiac stress. mTORC1 inhibition extends the life span of mice.13–15 It also reduces cardiac hypertrophy and improves cardiac function in the presence of pressure overload16–19 and genetic cardiomyopathies20–22 and reduces ischemic injury after acute23,24 and chronic myocardial infarction.19,25,26 Finally, the inhibition of mTORC1 reactivates cardiac autophagy, which is impaired in the presence of obesity and metabolic syndrome.23 These results are likely dependent on the fact that partial inhibition of mTORC1 eliminates the detrimental effects of the maladaptive functions of mTORC1 during cardiac stress, while maintaining its physiological functions. In this regard, it is known that rapalogs do not inhibit all the functions of mTORC1.27
Original received October 23, 2013; revision received November 28, 2013; accepted December 2, 2013. In November 2013, the average time from submission to first decision for all original research papers submitted to Circulation Research was 14.6 days. From the Cardiovascular Research Institute, Department of Cell Biology and Molecular Medicine, Rutgers New Jersey Medical School, Newark, NJ (S.S., J.S.); IRCCS Neuromed, Pozzilli, Italy (S.S., M.V.); and Division of Cardiology, Department of Clinical and Molecular Medicine, Faculty of Medicine and Psychology, University Sapienza, Rome, Italy (M.V.). Correspondence to Junichi Sadoshima, MD, PhD, Department of Cell Biology and Molecular Medicine, Rutgers New Jersey Medical School, 185 South Orange Ave, Medical Science Building G-609, Newark, NJ 07103. E-mail [email protected] © 2014 American Heart Association, Inc. Circulation Research is available at http://circres.ahajournals.org
DOI: 10.1161/CIRCRESAHA.114.302022
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550 Circulation Research January 31, 2014
Nonstandard Abbreviations and Acronyms 4E-BP1 Akt AMPK Atg eIF4E ERK GSK NF-κB PGC-1α PI3K PRAS40 Raptor Rheb Rictor S6K SGK-1 TAC TSC ULK
eIF4E-binding protein-1 protein kinase B adenosine monophosphate-activated protein kinase autophagy-related gene eukaryotic translation initiation factor 4E extracellular signal regulated kinase glycogen synthase kinase nuclear factor-κB peroxisome proliferator-activated receptor γ coactivator-1α phosphoinositide 3 kinase proline-rich Akt substrate 40 regulatory-associated protein of mammalian target of rapamycin (mTOR) Ras homolog enriched in brain rapamycin-insensitive companion of mTOR S6 kinase serum- and glucocorticoid-induced protein kinase-1 transverse aortic constriction tuberous sclerosis protein unc-51-like kinase
The degree of mTORC1 inhibition and the mTORC1 physiological functions needed to be preserved to convert mTORC1 inhibition from detrimental into beneficial during cardiac stress are unclear. In contrast, information about the pathophysiological functions of mTORC2 in the heart is still scarce. This article reviews and interprets the evidence currently available regarding the role of mTOR signaling pathway in cardiovascular physiology and disease (Table).
Biology of the mTOR Pathway The mTOR kinase is an atypical serine/threonine kinase of 289 kDa and belongs to the family of phosphoinositide
Figure 1. General cellular functions of mammalian target of rapamycin complex 1 (mTORC1) and mTORC2. The figure summarizes the most well-characterized functions of mTORC1 and mTORC2. mTORC1 regulates protein synthesis and autophagy in response to growth factors and stress. mTORC2 is known to regulate cell growth, survival, and polarity. Akt indicates protein kinase B; and AMPK, adenosine monophosphateactivated protein kinase.
3-kinase. It is encoded by a single gene in mammals, but it exerts its main cellular functions by forming mTORC1 and mTORC2 through assembly with specific adaptor proteins.4–8 mTORC1 and mTORC2 signaling pathways are evolutionarily conserved, and these represent the functional homologs of yeast TORC1 and TORC2. However, TOR1 and TOR2 are encoded by distinct genes in yeast, as first identified in Michael Hall’s laboratory in 1991.28 mTOR was isolated and cloned as a physical target of rapamycin through a cellular screening aimed at identifying the binding proteins of FK506-binding protein of 12 kDa–rapamycin complex.1–3 Rapamycin and its analogs bind to the cytosolic FK506-binding protein of 12 kDa, thereby forming a protein complex that only targets a specific domain of the mTOR protein when it is part of mTORC1. As a consequence of rapamycin–FK506-binding protein of 12 kDa binding, mTORC1 activity is strongly inhibited.4,6 Conversely, mTORC2 is relatively insensitive to rapamycin, although it has been demonstrated that prolonged treatment with rapamycin can also reduce the activity of mTORC2 by disrupting the complex.29 To date, proteins that are known to bind to mTOR in mTORC1 include regulatory-associated protein of mTOR (Raptor),30 mammalian lethal with sec-13 protein 8,31 proline-rich protein kinase B (Akt) substrate 40 (PRAS40),32 DEP domain-containing mTOR-interacting protein,33 and Tel two interacting protein 1/Tel2.34 Whereas, mTORC2 contains mTOR, the scaffold protein rapamycin-insensitive companion of mTOR (Rictor),35 stress-activated map kinase-interacting protein 1,36 protein observed with rictor,37 mammalian lethal with sec-13 protein 8,31 DEP domain-containing mTOR- interacting protein,33 and Tel two interacting protein 1/Tel2.34 The main functions of proteins forming the mTOR complexes are to maintain the integrity of the complexes, regulate their complex subcellular localization, and control their complex interactions with substrates and regulators4–8 (Figure 2). Evidence regarding the biological functions of mTORC1 and mTORC2 suggests that they have distinct cellular functions, substrates, and regulators.4–8 However, based on available evidence, it is difficult to distinguish the function of 1 complex completely from the other. It has been shown that the 2 complexes are functionally interconnected, and most of the studies that investigated the role of mTORC1 in the regulation of cellular homeostasis did not precisely rule out the involvement of mTORC2 in these mechanisms. In general, much more is known about the biology of mTORC1 than that of mTORC2 (Figure 2). mTORC1 plays a crucial role in the regulation of cellular homeostasis, growth, and response to stress. The main functions of mTORC1 are promotion of protein synthesis and, subsequently, cellular growth. The most studied substrates of mTORC1 are S6 kinase-1 (S6K1) and eukaryotic translation initiation factor 4E (eIF4E)-binding protein-1 (4E-BP1). mTORC1 phosphorylates and activates S6K1, which in turn promotes mRNA biogenesis and activates the protein translation process. In contrast, mTORC1 inhibits 4E-BP1 and allows the formation of the eIF4F complex that triggers cap-dependent translation.4–8 Additionally, mTORC1 promotes protein synthesis through the translation of 5′ terminal oligopyrimidine tract mRNAs and the promotion of ribosomal biogenesis.4–8 This mechanism explains why mTORC1 activity is often found to be increased in cancer cells.4–8
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Sciarretta et al mTOR Signaling in Cardiovascular System 551 Table. Main Studies That Investigated the Role of mTOR Kinase in the Regulation of Cardiac Physiology and Response to Stress In Vivo Cardiac Condition Development/ physiological conditions
Aging
Cardiac hypertrophy
Cardiac ischemia and reperfusion
Study
Type of mTOR Modulation and Animal Model
Effect on the Heart
Constitutive α-MHC-CRE–mediated mTOR gene deletion
High embryonic lethality due to cardiac failure, massive cardiac dilation, dysfunction, heart failure, early mortality in the postnatal stage, and metabolic derangements
Zhang et al10
Tamoxifen-induced α-MHC-CRE–mediated mTOR deletion during adulthood
Massive cardiac dilation, apoptosis, autophagy, mitochondrial abnormalities, sarcomere disarray, cardiac dysfunction, heart failure, and early mortality
Shende et al11
Tamoxifen-induced α-MHC-CRE–mediated mTOR deletion during adulthood
Massive cardiac dilation, apoptosis, autophagy, mitochondrial abnormalities, sarcomere disarray, metabolic abnormalities, cardiac dysfunction, heart failure, and early mortality
Tamai et al12
Constitutive α-MHC-CRE–mediated rheb1 gene deletion
Reduction of mTORC1 activity 5 days after birth, defect in physiological cardiac growth, cardiac dilation, dysfunction, heart failure, mortality ≤10 days after birth
Flynn et al14
Pharmacological mTOR inhibition using rapamycin
Reduced age-induced cardiac inflammation and fibrosis, upregulation of energy metabolism
Zhou et al80
Systemic GSK-3α deletion
Cardiac hypertrophy, dysfunction, and sarcomere abnormalities during aging through mTOR activation and autophagy inhibition
Zhang et al10
Tamoxifen-induced α-MHC-CRE–mediated mTOR deletion during adulthood and pressure overload
Inhibition of compensatory hypertrophy, inhibition of protein synthesis, rapid cardiac dilation, apoptosis, cardiac dysfunction, and heart failure
Shende et al11
Tamoxifen-induced α-MHC-CRE–mediated mTOR deletion during adulthood and pressure overload
Inhibition of compensatory hypertrophy, inhibition of protein synthesis, rapid cardiac dilation, cardiac dysfunction, and heart failure
Shioi et al16
mTORC1 inhibition using rapamycin and then pressure overload
Inhibition of pathological cardiac hypertrophy and preservation of cardiac function
McMullen et al17
mTORC1 inhibition using rapamycin in mice with established TAC-induced cardiac hypertrophy
Regression of compensated and decompensated hypertrophy, improved cardiac function in the latter
Völkers et al18
AAV9-mediated PRAS40 overexpression in hearts subjected to pressure overload
Prevention of TAC-induced pathological hypertrophy and myocardial fibrosis, maintenance of cardiac function
Wu et al19
Constitutive α-MHC-CRE–mediated heterozygous rheb1 gene deletion and then pressure overload
Reduction of TAC-induced hypertrophy and fibrosis
Marin et al20
Mouse model of LEOPARD disease treated with rapamycin
Regression of hypertrophic cardiomyopathy and cardiac disarray
Choi et al22; Ramos et al21
Mouse model of cardiomyopathy caused by Lamin A/C mutation treated with pharmacological mTORC1 inhibition
Autophagy reactivation and improved cardiac function, muscle dystrophy, and increased survival
Song et al99
α-MHC-CRE–mediated mTOR overexpression
No increase in cardiac mass, preservation of cardiac function and reduced myocardial inflammation through inhibition of NF-κB signaling
Sciarretta et al23
Pharmacological inhibition of mTORC1 during prolonged ischemia, prolonged ischemia in mice with α-MHC-CRE–mediated Rheb overexpression
Inhibition of ischemia-induced mTORC1 downregulation and autophagy activation, increased infarct size after ischemia that is reversed by rapamycin treatment
Zhai et al24
Mice with α-MHC-CRE–mediated dominant negative GSK-3β overexpression and systemic heterozygous GSK-3β deletion subjected to prolonged ischemia and I/R
GSK-3β is activated during prolonged ischemia but inhibited during reperfusion; genetic inhibition of GSK-3β increases ischemic injury after prolonged ischemia but reduces reperfusion injury through a deregulated activation of mTORC1
Buss et al25
mTORC1 inhibition with everolimus during chronic myocardial infarction
Reduction of infarct size, pathological growth, and cardiac dilation; improvement of cardiac function; activation of autophagy
AAV9-mediated PRAS40 cardiac overexpression and Rictor knockdown in mice subjected to chronic myocardial infarction
Overexpression of PRAS40 inhibits mTORC1, reduces ischemic injury, apoptosis and cardiac remodeling, and improves cardiac function in an mTORC2-dependent manner; Rictor knockdown causes deterioration of cardiac function and remodeling after myocardial infarction
Di et al111
Rapamycin and S6K inhibitors during chronic myocardial infarction
Reduction of cardiac ischemic remodeling and cardiomyocte apoptosis
Aoyagi et al119
α-MHC-CRE–mediated mTOR overexpression and I/R
Inhibition of cardiac remodeling after I/R in vivo, and reduction of necrosis and myocardial inflammation after I/R ex vivo (Continued )
Zhu et al
9
Völkers et al26
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552 Circulation Research January 31, 2014 Table. Continued Cardiac Condition Metabolic disorders
Study Wang et al
Type of mTOR Modulation and Animal Model
Effect on the Heart
Rapamycin treatment during high-fat diet– induced obesity
Akt and mTOR are activated in the vasculature of obese animals; rapamycin inhibits endothelial senescence and the susceptibility to peripheral ischemia in obese animals
Sciarretta et al23
Rapamycin treatment and partial inducible mTOR genetic disruption in mice with high-fat diet–induced obesity and metabolic syndrome subjected to prolonged ischemia
Ischemia-induced autophagy activation is inhibited in the hearts of obese animals through deregulated activation of Rheb and mTORC1; infarct size after ischemia is larger in obese animals; pharmacological and partial genetic inhibition of mTOR rescue autophagy and reduce ischemic injury in obese mice
Li et al126
Swine model of metabolic syndrome
Increased cardiac mTOR level and reduction of cardiac autophagy that is proportional to the degree of derangements of cardiac structure and function
Guo et al127
Rapamycin in mice with high-fat diet–induced obesity
Increased mTOR activity and reduced autophagosome formation in the hearts of obese mice, associated with a reduction of cardiac function; cardiac function is rescued by rapamycin and deteriorated by genetic adiponectin disruption
Xu et al128
Mice with systemic Akt2 knockout subjected to high-fat diet
mTOR activation and disruption of autophagic flux in the hearts of obese mice; Akt2 deletion rescues autophagic flux and cardiac dysfunction in obese animals
Xu et al129
Streptozotocin-induced diabetes mellitus
Cardiac autophagosome formation and flux are impaired in diabetic mice; these effects are associated with increased mTOR activity
124
AAV9 indicates adeno-associated virus 9; Akt, protein kinase B; GSK-3β, glycogen synthase kinase-3β; I/R, ischemia/reperfusion; LEOPARD, Lentigines, ECG conduction abnormalities, Ocular hypertelorism, Pulmonic stenosis, Abnormal genitalia, Retardation of growth and sensorineural Deafness; mTOR, mammalian target of rapamycin; mTORC1, mTOR complex 1; NF-κB, nuclear factor-κB; PRAS40, proline-rich Akt substrate 40; Rheb, Ras homolog enriched in brain; Rictor, rapamycininsensitive companion of mTOR; S6K, S6 kinase; and TAC, transverse aortic constriction.
mTORC1 also promotes cell proliferation by promoting the de novo synthesis of cellular membrane lipids through the sterol regulatory element-binding protein 1/2–dependent expression of lipogenic genes.7,38 However, mTORC1 not only activates anabolic processes but also inhibits catabolic processes. mTORC1 strongly inhibits autophagy, an evolutionarily conserved intracellular bulk degradation process responsible for cellular degradation of proteins and organelles.6,7,39,40 mTORC1 seems to regulate autophagy both at post-translational and transcriptional levels. It phosphorylates the autophagic protein unc-51-like kinase (ULK) 1/2, thereby inhibiting the macrocomplex ULK1/autophagy-related gene (Atg) 13/focal adhesion kinase family interacting protein-200 that promotes autophagosome formation.41 Additionally, mTORC1 activation inhibits the expression of autophagic proteins, particularly Atg7,23 which is crucial for the initiation of the autophagic process.39 It has been shown that mTOR significantly inhibits the p73 factor and the transcription factor EB that can induce autophagy through the upregulation of autophagic proteins such as Atg7.42–44 mTORC1 also plays an important role in the regulation of cellular metabolism. mTORC1 promotes mitochondrial biogenesis and oxidative metabolism through the peroxisome proliferator–activated receptor γ coactivator-1α (PGC-1α)-mediated activation of the transcription factor Ying-Yang 1.45 mTORC1 can activate glycolysis and the oxidative pentose phosphate pathway through the activation of hypoxia-inducible factor-1α and sterol regulatory element–binding protein 1/2, respectively.46 Finally, mTORC1 activity is inhibited by acid deprivation, energy stress, hypoxia, ER stress, and genotoxic stress.4–8 Under these conditions, mTORC1 inhibition allows the upregulation
of stress responsive mechanisms that limit cell death, such as reduction of protein synthesis, autophagy, cell cycle arrest, and DNA repair. The Akt and adenosine monophosphate–activated protein kinase (AMPK) pathways represent the most well- characterized regulators of mTORC1. In the presence of nutrients and growth factors such as insulin, insulin growth factor-1, platelet-derived growth factor, and epidermal growth factor, Akt is activated and, in turn, phosphorylates and activates mTOR or inhibits PRAS40, an endogenous mTORC1 inhibitor.4–8,32,47 Whereas, AMPK, which is inactive in the presence of nutrients and high ATP levels, is strongly activated during energy deprivation and other types of cellular stress. AMPK activates the tuberous sclerosis protein (TSC) 1/TSC2 complex,48 which displays a strong GTPase activity and inhibits the small GTP-binding protein Ras homolog enriched in brain (Rheb), a direct mTORC1 activator.49,50 Glycogen synthase kinase (GSK) 3β can also activate the TSC1/ TSC2 complex and inhibit mTORC1 during energy stress.48 Alternatively, regulated in development and DNA damage response 1, which is upregulated during hypoxia, activates TSC2 independently of AMPK and GSK-3β.51 Rheb can also be inhibited by p38-regulated/activated kinase during energy deprivation, independently of TSC2.52 Conversely, the Akt, extracellular signal regulated kinase (ERK) 1/2, and inhibitor of NF-κB kinase-β pathways inhibit the TSC1/TSC2 complex in response to growth factors and cytokines.53–55 Recently, a new mechanism promoting the activation of mTORC1 has been elucidated. In the presence of amino acids, Rag GTPases are activated and mediate mTORC1 translocation to lysosome membranes, where mTORC1 is activated by Rheb.56,57
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Sciarretta et al mTOR Signaling in Cardiovascular System 553
Figure 2. General overview of the mammalian target of rapamycin (mTOR) signaling pathway. The dashed line signifies that rapamycin inhibits mTOR complex 2 (mTORC2) in specific cell types or after prolonged treatment. 4EB-P1 indicates eukaryotic translation initiation factor 4E (eIF4E)-binding protein-1; Akt, protein kinase B; AMPK, adenosine monophosphate-activated protein kinase; Atg7, autophagy-related gene 7; DEPTOR, DEP domain-containing mTOR-interacting protein; ERK1/2, extracellular signal regulated kinase 1/2; FKBP12, FK506-binding protein of 12 kDa; GSK-3β, glycogen synthase kinase-3β; HIF-1, hypoxia-inducible factor-1α; IKKβ, inhibitor of NF-κB kinase-β; mLST8, mammalian lethal with sec-13 protein 8; PGC-1α, peroxisome proliferator-activated receptor γ coactivator-1α; PI3K, phosphoinositide 3 kinase; PKC-α, protein kinase C-α; PRAK, p38-regulated/activated kinase; PRAS40, proline-rich Akt substrate 40; PROTOR, protein observed with rictor; Raptor, regulatory-associated protein of mTOR; REDD1, regulated in development and DNA damage response 1; Rheb, Ras homolog enriched in brain; RhoA, Ras homolog gene family, member A; S6K1, S6 kinase-1; SGK1, serum- and glucocorticoid-induced protein kinase-1; SREBP1/2, sterol regulatory element-binding protein 1/2; TFEB, transcription factor EB; TSC1/2, tuberous sclerosis protein 1/2; Tt1, Tel two interacting protein 1; ULK1/2, unc-51-like kinase 1/2; and YY1, Ying-Yang 1.
This process seems to be negatively regulated by microtubuleassociated protein/microtubule affinity-regulating kinase 4, independently of Rheb activity.58 mTORC2 can be activated by insulin and growth factors, whereas it is relatively insensitive to nutrient deprivation.5,6 The phosphoinositide 3 kinase (PI3K) pathway was shown to activate mTORC2,59 and it seems that the TSC1/2 complex does as well.60 mTORC1 seems to inhibit mTORC2 through phosphorylation of Rictor,61 suggesting that mTORC1 and mTORC2 are functionally interconnected. This hypothesis is also strongly supported by the fact that the best known substrate of mTORC2 is Akt, which is phosphorylated at serine 473, particularly in response to insulin.62,63 The biological importance of mTORC2-dependent phosphorylation of Akt is not yet understood, because it has been shown that mTORC2 is dispensable for Akt phosphorylation in certain cell types and conditions.64 Both protein kinase C-α65 and serum- and glucocorticoid-induced protein kinase-1 (SGK-1)66 have been shown to be substrates of mTORC2, despite mTORC1 also being shown to regulate SGK-1.67 Whether SGK-1 exerts different functions in cardiomyocytes when it is activated by mTORC1 or mTORC2 is unclear. The most studied function
of mTORC2 is the regulation of survival and growth, likely through the regulation of Akt and SGK-1. SGK-1 has been shown to promote cardiomyocyte survival while inhibiting hypertrophy,68 whereas SGK-1 chronic activation during heart failure is detrimental.69 mTORC2 also regulates cell polarity and cytoskeletal organization through the regulation of protein kinase C-α and Ras homolog gene family member A.65 Protein kinase C-α has also been shown to negatively regulate cardiac contractility.70
Role of mTOR Signaling in the Regulation of Cardiac Homeostasis and Physiological Growth Given its myriad cellular functions, it is not surprising that mTOR kinase is necessary for normal regulation of cardiomyocyte homeostasis and growth during both development and postnatal period (Figure 3). Systemic mTOR- and Raptor- knockout embryos die early during development, right after implantation.63 However, embryos with constitutive α-MHCCre–mediated mTOR deletion also display a dramatic mortality starting around E13.5, with only 8% of embryos surviving the developmental stage. Cardiac-specific mTOR-knockout embryos present a significant reduction in cardiomyocyte
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554 Circulation Research January 31, 2014 proliferation and an increase in apoptosis, with a 34% reduction in cardiomyocyte number. As a result, cardiac-specific mTOR-knockout embryos present cardiac dilation and dysfunction, with signs of terminal cardiac failure.9 Consistent with these results, systemic deletion of the rheb1 gene that extensively reduces mTORC1 activity is embryonically lethal, most likely because of cardiac defects such as ventricular wall thinning and cardiomyocyte apoptosis.71 Global rictor gene deletion that selectively disrupts mTORC2 is also lethal in the developmental stage, and embryos with rictor deletion display significant cardiovascular abnormalities.35 Thus, both mTORC1 and mTORC2 are highly important for cardiac development and embryo survival. The mTOR pathway also seems vital for the maintenance of cardiac structure and function in the postnatal period and adulthood. Mice with α-MHC-Cre–mediated cardiac mTOR disruption that are born alive die within a few weeks after birth from massive cardiac dilation, dysfunction, and heart failure.9 mTOR-knockout mice present with derangements in fatty acid metabolism in the heart. Inducible cardiac-specific mTOR deletion in adulthood also leads to cardiac dysfunction and heart failure, with chamber dilation and wall thinning. mTOR-knockout mice display massive apoptosis, fibrosis, autophagy, mitochondrial abnormalities and dysfunction, sarcomere disarray, and ultimately death
