Cardiac-specific Raptor ablation revealed that the mTOR pathway is essential during early embryonic

To investigate the role of the mTOR signaling pathway during early cardiac development and specifically within the heart excluding secondary effects, we generated mice with cardiac-specific Raptor ablation. The RAPTOR protein directly interacts with MTOR and is essential for mTORC1 assembly and function^120,121. Genetic Raptor ablation causes disassembly of the mTORC1 complex, consequently impairing downstream signaling²⁰⁶.

Cardiac-specific Raptor ablated mice died in utero between 10.5 dpc and 11.5 dpc (see Chapter 6.4.1, Figure 16A), suggesting that the mTOR pathway is essential during early heart development and thus for survival of the entire organism. Analyses of cardiac morphology revealed a fulminant phenotype at 10.5 dpc with severely reduced ventricular wall thickness, resulting in hypoplastic hearts (see Chapter 6.4.2, Figure 16E). Recent unpublished data from our lab demonstrated a significantly reduced proliferative potential of cardiomyocytes in 10.5 dpc cRaptor^-/- hearts. Other mouse models with similar phenotypes and proliferation defects support our findings. For instance, mice lacking all three D-type cyclins (Ccnd1, Ccnd2, Ccnd3) die around 16.5 dpc due to heart abnormalities and impaired proliferation of myocardial cells²⁶⁷. Fetal cardiomyocyte-restricted inactivation of the yes-associated protein 1 (Yap1) gene also causes decreased cardiomyocyte proliferation leading to myocardial hypoplasia and embryonic lethality after 16.5 dpc⁵⁹. Moreover, we argued that in neonatal rapamycin treated hearts increased apoptosis is mediated by unrestricted autophagy (see Chapter 7.1.3). A similar scenario might contribute to hypoplasia in cRaptor -/-hearts. We assume that reduced proliferation and potentially increased apoptosis result in a reduced set of cardiac cells in 10.5 dpc cRaptor^-/- hearts, causing the severe thinning of both the compact as well as the trabeculated myocardium. Hence, future attention should be paid on cellular and molecular mechanisms involved in autophagy and apoptosis in the

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specific Raptor KO model. The trabeculae, which develop between 8.5 dpc and 10.5 dpc, are especially important for proper and powerful contraction as well as for nutrient uptake from the ventricular blood flow by increasing the surface area, as a coronary artery system to supply the myocardium has not been developed at that stage (see Chapter 3.3.1).

Consequently, a reduced set of contracting cardiac cells in combination with the almost absent trabeculae in cRaptor^-/- hearts might result in insufficient nutritional supply of the growing myocardium and in the end cause death of the entire embryo.

Interestingly, embryonic cMtor-KO mice die between 13.5 dpc and the end of gestation¹⁹¹. In these mice, cardiac-specific Mtor KO was achieved by using a myosin heavy chain-driven Cre recombinase¹⁹¹. However, only a subset of cardiomyocytes displays Mtor inactivation, because mosaic expression of Cre recombinase was observed¹⁹¹. Therefore, the embryonic day at which cMtor-KO mice die was shown to be dependent on the extent of cardiac Cre expression and hence Mtor inactivation¹⁹¹. Consequently, the reason for the longer survival of cMtor-KO embryos compared to our cRaptor^-/- mice might be the cardiomyocyte mosaic for the KO of Mtor. Interestingly, cardiac proliferation rates in 10.5 dpc and 12.5 dpc cMtor-KO embryos were found to be significantly reduced¹⁹¹; a finding that is consistent with our cRaptor^-/- model.

In line, prenatal rapamycin treatment from 11.5 dpc onwards causes fetal lethality (see Chapter 6.3.1 and Supplementary Figure 4), as demonstrated by a pilot experiment which was terminated after assessing the extensive consequences for the offspring. Importantly, mTORC1 inhibition by rapamycin during late gestation from 15.5 dpc does not result in prenatal lethality (see Chapter 6.3.2, Figure 10), even though neonatal rapamycin treated hearts are hypoplastic (see Chapter 6.3.3.1, Figure 11C-E). Analyses of neonatal cardiac proliferation rates revealed no significant differences between prenatal vehicle and rapamycin treatment (see Chapter 6.3.3.2, Figure 12C). However, proliferation was assessed using immunofluorescence staining for KI67, which labels proliferating cells in all active phases of the cell cycle²¹⁷. Thus, a more specific proliferation marker, such as p-HH3, which is restricted to mitosis and the late G2 phase of the cell cycle²¹⁸, might help to uncover subtle differences in proliferation rates in the neonatal stage. Importantly, differences in survival as well as in proliferation rates between the above discussed approaches are most likely due to the different phases of development during which the mTOR pathway was inhibited.

Moreover, cell cycle activity in peri- and postnatal cardiomyocytes does not necessarily mean cell division⁶⁹. It is conceivable to speculate that in controls, cardiomyocytes still actively divide, whereas they start to become binucleated in cHccs^+/- neonates. In order to focus on potential differences regarding the moment when terminal differentiation occurs, cytokinesis markers, such as aurora B kinase (a chromosomal passenger protein that is involved in chromosome segregation, spindle-checkpoint, and cytokinesis)²⁶⁸ or determination of the

DISCUSSION

actual cell number might prove useful. Additionally, analyzing cardiomyocyte proliferation in fetal rapamycin treated hearts shortly before birth (e.g. between 17.5 dpc and 18.5 dpc) might help to reliably elucidate the impact of rapamycin treatment on cardiomyocyte proliferation rates. In this context, the IUGR phenotype of rapamycin treated neonatal hearts might not only be caused by reduced cardiomyocyte size and increased apoptosis rates, as discussed above, but also by reduced proliferation and thus reduced number of cardiac cells.

This would again correlate with findings in classical IUGR rat models at birth: less cardiomyocytes²³ as well as a suppressed replicative potential of cardiac cells upon intrauterine LPD²⁴ and decreased cardiomyocyte number after placental insufficiency³³. However, one can also speculate that the mTOR pathway plays different roles during different phases of cardiac development. In this context, our results suggest that during embryonic development, the mTOR signaling cascade regulates proliferation; while in later stages it might control cell size. Similar mechanisms have been proposed for cell cycle regulating genes. For instance certain cyclins were shown to be associated with proliferation in embryonic but with hypertrophy in adult cardiomyocytes76,77,231,269,270. Moreover, mTOR pathway hyperactivation is often associated with tumor growth, where it certainly promotes proliferation^271-273. Hence, it appears that the same growth machinery (mTOR pathway) can induce proliferation in one cell type but increases cell size in the other. The latter seems to primarily affect terminally differentiated cardiomyocytes. Thus, for the heart, as long as cardiomyocytes are in principle able to divide, the mTOR pathway might induce proliferation, but as soon as cardiomyocytes withdraw from the cell cycle the mTOR signaling cascade might induce hypertrophy. This consideration would make the mTOR pathway especially interesting with regard to a potential role in the transition from hyperplastic to hypertrophic cardiac growth in the perinatal period, given that until now the molecular players initiating this switch are only insufficiently elucidated (see Chapter 3.3.2).

Strikingly, homozygous cardiac-specific Raptor ablation completely abolishes phosphorylation of 4E-BP1 in 10.5 dpc embryonic hearts (see Chapter 6.4.3, Figure 16D+F).

This is in line with cardiac-specific Mtor or Raptor ablation in adulthood as well as with 12.5 dpc embryonic cMtor-KO mice182,183,191. In all three models significantly decreased cardiac 4E-BP1 phosphorylation was observed182,183,191. In contrast, mTORC1 inhibition by prenatal rapamycin treatment severely inhibited phosphorylation of S6K1 and S6 but not of 4E-BP1 in neonatal hearts (see Chapter 6.3.1, Figure 9A). Differential effects of rapamycin on phosphorylation of various mTORC1 downstream targets have previously been reported in cell culture²⁷⁴. In this regard, it was demonstrated that rapamycin potently inhibits S6K1 activity throughout the duration of treatment, while 4E-BP1 phosphorylation recovers within a few hours despite continued rapamycin exposure²⁷⁴. Phosphorylated 4E-BP1 dissociates from EIF4E, allowing EIF4E to bind to other translation initiation factors to initiate

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dependent translation¹⁶². Thereby, EIF4E specifically enhances the translation of transcripts, which often encode proteins associated with a proliferative response, such as CYCLIN D1²⁷⁵. Moreover, recent studies revealed that the sequence composition of mTORC1 phosphorylation sites is one of the key determinants of whether the site is a good or a poor mTORC1 substrate²⁷⁶. In this context, differences in substrate quality were shown to be most important to allow downstream effectors of mTORC1 to respond differentially to temporal and intensity changes in the levels of nutrients and growth factors or pharmaceutical inhibitors such as rapamycin²⁷⁶. Furthermore, cell culture experiments demonstrated that Eif4e overexpression is associated with significant acceleration of G1 phase progression from G0 phase to S phase, indicating cell cycle control²⁷⁷). Thus, it is tempting to speculate that the differences in 4E-BP1 phosphorylation are at least partially responsible (next to the developmental stage as well as differences in the proliferative potential of 10.5 dpc and P1 cardiomyocytes) for the different effects of cardiac-specific Raptor ablation and rapamycin treatment, respectively, on cell proliferation.

Taken together, all these findings indicate that regular cardiac mTOR pathway activity is essential during embryonic development, given that mTOR signaling appears to be required for normal cardiomyocyte proliferation and thus for the survival of the entire organism.