Rheb1Plays A Vital Role In Heart Growth And Function Maintenance In Postnatal Mice

Background:Heart failure in infant is a severe health problem worldwide. During infant, dramatic growth and remodeling is one of the significant features of heart development. Disruption of heart growth and remodeling during postnatal stage results in heart hypertrophy or hypotrophy, eventually leading to heart failure. Protein kinase B (PKB/Akt) signaling plays a vital role in postnatal heart growth. Mammalian target of rapamycin complex1(mTORC1) downstream of Akt controls protein translation, energy metabolism, apoptosis and autophagy. Ras homologue enriched in brain1(Rheb1) upstream of mTORC1belongs to small GTPase Ras superfamily. Previous studie showed that Rhebl-mTORC1signaling regulates physiological hypertrophy in rat cardiomyocyte. Also, our previous study showed Rheb1-mTORC1signaling is involved in pathologically cardiac remodeling in mice. However, the roles of Rhebl in postnatal heart development are still elusive.Objectives:To explore the roles of Rheb1in postnatal heart growth and physiological remodeling.Methods:Using cadiac specific knockout technique, we deleted Rheb1in postnatal cardiomycyte in mice. We further observed morphologic changes of Rheb1-deletion heart by gross anatomy, hematoxylin and eosin (H&E) staining, Masson’s staining and electron microscope and measured the physiological parameters body weight, heart weight and the ratio of heart weight to body weight. Isolated cardiomyocytes were also observed and their surface areas were measured. By echocardiography and electrocardiography, heart function and electrical activity were detected. Furthermore, we detected mTORC1signaling pathway, Akt activity, autophagic markers, apoptotic markers and metabolism-related gene profiles in order to explore the molecular mechanisms of Rhebl-deletion induced cardiac phenotypes. At last, we evaluated the role of Akt activation by releasing negative feedback inhibition in survival of Rheb1knockout mice through removal of phosphatase and tensin homolog (Pten).Results:Rhebl gene was deleted partially at postnatal day3and nearly totally deleted at day5to7using mTmG cre report mice. Western blot analyzing showed Rheb1protein levels were significantly decreased at postnatal day7compared with control group. Rheb1-deletion mice were born in Mendelian ratio but started to die11days after birth. All of these mice were lost by16days. At day9after birth, heart growth was retarded and heart-to-body weight ratio decreased in Rheb1-deletion mice compared to control group. Isolated cardiomyocyte surface areas were significantly smaller in the mutant than that in the control from postnatal day9; meanwhile cardiomyocyte numbers were decreased by wheat germ agglutinin (WGA) staining analysis. Body weight was lower in the mutant than in the control1-2days before death. Echocardiographic analysis revealed that Rhebl-deficient mice exhibited cardiac dilatation and reduced contractility. Electrocardiography recordings showed intermittent complete heart block and sinus arrest as well as ventricular arrhythmia in the mutant. Moreover, TUNEL staining and cleaved caspase3staining showed that apoptotic cardiomyocytes increased before heart failure in Rheb1-deficient mice compared with the control. Unexpectedly, autophagy marker LC3Ⅱ was not increased while LC3Ⅰ was significantly decreased in the mutant compared to the control group. In order to explore the mechanisms of phenotypes in Rhebl mutant mice, we studied the activity of mTORC1signaling, the changes of energy metabolism-related gene profiles, apoptosis-related signaling pathway. Compared with the control group, the phosphorylation levels of both ribosomal protein6(S6) and eukaryotic initiation factor4E-binding protein1(4E-BP1), the downstream targets of mTORC1signaling, were dramatically reduced in the mutant. Thus, we concluded that Rhebl is indispensible for activation of mTORCl signaling in mammalian postnatal hearts and that Rhebl-mTORCl signaling is essential to regulate heart growth and function maintenance. In additional, glucose and fatty acid metabolisms were compromised. Intriguingly, we found that caspase12, C/EBP homologue protein (CHOP) and phospho-c-Jun N-terminal kinase (JNK) signaling were upregulated significantly in the mutant compared with the control. Furthermore, the activating transcription factor6(ATF6) and ribonuclease inositol-requiring protein-1(IRE1) but not double-stranded RNA-activated protein kinase-like ER kinase (PERK), the key mediators of endoplasmic reticulum (ER) stress, were activated in Rheb1-deficient mice compared with the control group. As releasing feedback of insulin receptor substrate1(IRS1) by S6, Akt activity was significantly increased in the mutant compared to the wild type. To elucidate the effect of increased Akt activity in this context, we respectively generated Rhebl/Aktl and Rhebl/Pten double knockout mice to decrease and increase Akt activity further. By survival analysis, Rhebl/Pten double knockout (dKO) mice lived significantly longer than Rhebl knockout mice whereas Rhebl/Aktl dKO lived shorter.Conclusions:This study has demonstrated that Rhebl is essential to mTORCl activation and physiological heart growth as well as maintenance of heart function and electrophysiological homeostasis in postnatal mice. There is a crosstalk between Rheb1-mTORC1signaling and ER stress-induced apoptotic pathways which regulate postnatal cardiomyQcytes survival. Non-mTORCl signaling may contribute to Rhebl deletion induced cardiomyocyte autophagy in postnatal mice. Activation of Akt may be beneficial to survival in mice with heart failure.