Mechanistic Target of Rapamycin (Mtor) Is Essential for Murine Embryonic Heart Development and Growth Yi Zhu 1,2 , Karla M. P. Pires 1,4 , Kevin J. Whitehead 3 , Curtis D. Olsen 3 , Benjamin Wayment 3 , Yi Cheng Zhang 1 , Heiko Bugger 1¤a , Olesya Ilkun 1 , Sheldon E. Litwin 3¤b , George Thomas 5 , Sara C. Kozma 5 , E. Dale Abel 1,2 * 1 Division of Endocrinology, Metabolism, and Diabetes and Program in Molecular Medicine, School of Medicine, University of Utah, Salt Lake City, Utah, United States of America, 2 Department of Biochemistry, School of Medicine, University of Utah, Salt Lake City, Utah, United States of America, 3 Division of Cardiology, School of Medicine, University of Utah, Salt Lake City, Utah, United States of America, 4 Biomedical Center, Institute of Biology, Laboratory of Morphometry and Cardiovascular Morphology, State University of Rio de Janeiro, Rio de Janeiro, Brazil, 5 Division of Hematology-Oncology, Department of Internal Medicine, Metabolic Diseases Institute, College of Medicine, University of Cincinnati, Cincinnati, Ohio, United States of America Abstract Mechanistic target of rapamycin (Mtor) is required for embryonic inner cell mass proliferation during early development. However, Mtor expression levels are very low in the mouse heart during embryogenesis. To determine if Mtor plays a role during mouse cardiac development, cardiomyocyte specific Mtor deletion was achieved using a myosin heavy chain (a- MHC) driven Cre recombinase. Initial mosaic expression of Cre between embryonic day (E) 10.5 and E11.5 eliminated a subset of cardiomyocytes with high Cre activity by apoptosis and reduced overall cardiac proliferative capacity. The remaining cardiomyocytes proliferated and expanded normally. However loss of 50% of cardiomyocytes defined a threshold that impairs the ability of the embryonic heart to sustain the embryo’s circulatory requirements. As a result 92% of embryos with cardiomyocyte Mtor deficiency died by the end of gestation. Thus Mtor is required for survival and proliferation of cardiomyocytes in the developing heart. Citation: Zhu Y, Pires KMP, Whitehead KJ, Olsen CD, Wayment B, et al. (2013) Mechanistic Target of Rapamycin (Mtor) Is Essential for Murine Embryonic Heart Development and Growth. PLoS ONE 8(1): e54221. doi:10.1371/journal.pone.0054221 Editor: Moises Mallo, Instituto Gulbenkian de Cie ˆ ncia, Portugal Received July 17, 2012; Accepted December 10, 2012; Published January 14, 2013 Copyright: ß 2013 Zhu et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: EDA was funded by U01HL087947, R01DK092065. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: dale.abel@hmbg.utah.edu ¤a Current address: Division of Cardiology, University of Freiburg, Freiburg im Breisgau, Germany ¤b Current address: Division of Cardiology, Georgia Health Sciences University, Augusta, Georgia, United States of America Introduction Mechanistic target of rapamycin (Mtor) engages in two distinct complexes (MtorC1 and MtorC2) to integrate both intracellular and extracellular signals through multiple cellular pathways to regulate cell metabolism, growth, function and survival [1]. Germline disruption of Mtor in mice leads to early embryonic lethality due to impaired proliferation and G1 arrest of embryonic stem cells [2,3]. MtorC1 comprises Mtor, a scaffold protein raptor, the proline- rich Akt substrate of 40KD (PRAS40) and the LST8 homolog (Mlst8) [4]. MtorC1 regulates protein translation through the direct phosphorylation of S6K1 and 4E-BP1 proteins. Deletion of raptor (MtorC1) in mouse skeletal muscle results in muscle atrophy and decreased muscle function [5]. Deletion of raptor (MtorC1) in mouse adipocytes results in reduced size of the adipose depot, and mice are protected against diet-induced obesity and hypercholes- terolemia as a result of increased mitochondrial uncoupling in adipocytes [6]. MtorC2 comprises Mtor, a scaffold protein rictor, hSin1, PRAS40 and Mlst8. MtorC2 was shown to act as a PDK2, which phosphorylates the Serine-Threonine kinase Akt/PKB on the Ser473 residue [7]. However, phosphorylation of Akt/PKB at Ser473 only affects Akt/PKB’s kinase activity toward a subset of downstream targets such as members of the forkhead family of transcription factors (FOXOs) [8,9]. Tissue specific disruption of MtorC2 by rictor deletion leads to mild effects [10]. For example, mice with rictor deletion in skeletal muscle appear normal [5], and mice with rictor deletion in adipose tissue results in bigger mice due to increased whole body insulin and IGF1 levels, but the adipose tissue size was not changed [11]. Moreover MtorC2 might not be the sole PDK2: as deletion of both raptor and rictor in skeletal muscle results in elevated Akt/PKB Ser473 phosphoryla- tion [5]. Skeletal muscle specific Mtor deletion phenocopies raptor deletion, indirectly suggesting that MtorC2 may play a minor role in skeletal muscle [12]. In the heart, MtorC1 is an important modulator of Akt/PKB regulated cardiac hypertrophy, and rapamycin treatment was able to prevent the hypertrophy induced by overexpressing a consti- tutively activated Akt1 [13]. However, cardiac specific overex- pression of constitutively activated Mtor does not increase heart weight significantly [14]. By contrast, inducible deletion of Mtor in cardiomyocytes leads to heart failure and demise of the mouse on the basis of induction of 4E-BP1 protein, which binds to eukaryotic initiation factor 4E (eIF4E) and shuts down cap-dependent protein translation in cells [15]. The report also showed that whole body PLOS ONE | www.plosone.org 1 January 2013 | Volume 8 | Issue 1 | e54221