Cardiac restricted overexpression of kinase-dead mammalian target of rapamycin (mTOR) mutant impairs the mTOR-mediated signaling and cardiac function

Abstract

Mammalian target of rapamycin (mTOR) is a key regulator for cell growth through modulating components of the translation machinery. Previously, numerous pharmacological studies using rapamycin suggested that mTOR has an important role in regulating cardiac hypertrophic growth. To further investigate this assumption, we have generated two lines of cardiac specific mTOR transgenic mice, kinase-dead (kd) mTOR and constitutively active (ca) mTOR, using alpha-myosin heavy chain promoter. alpha-Myosin heavy chain (alphaMHC)-mTORkd mice had a near complete inhibition of p70 S6k and 4E-BP1 phosphorylation, whereas alphaMHC-mTORca had a significant increase in p70 S6k and 4E-BP1 phosphorylation. Although the cardiac function of alphaMHC-mTORkd mice was significantly altered, the cardiac morphology of these transgenic mice was normal. The cardiac hypertrophic growth in response to physiological and pathological stimuli was not different in alphaMHC-mTORkd and alphaMHC-mTORca transgenic mice when compared with that of nontransgenic littermates. These findings suggest that the mTOR-mediated signaling pathway is not essential to cardiac hypertrophic growth but is involved in regulating cardiac function. Additional analysis of cardiac responses to fasting-refeeding or acute insulin administration indicated that alphaMHC-mTORkd mice had a largely impaired physiological response to nutrient energy supply and insulin stimulation.

FIGURE 1.

Generation of αMHC-mTOR^kd and αMHC-mTOR^kd transgenic mice. A, schematic diagram of constructs. B, Western blot analysis of the level of transgenic mTOR expression, total mTOR, phospho-S6, phospho-Akt, and different phosphorylated 4E-BP1 isoforms in αMHC-mTOR^kd and αMHC-mTOR^ca transgenic mice and littermate controls. Cardiac protein extracts were probed with antibodies against AU1 tag, mTOR, phospho-S6, 4E-BP1, and eIF4E (loading control). 4E-BP1 phosphorylation was demonstrated by changes in the migration rate of the protein on 15% SDS-PAGE, with increased phosphorylation (γ) corresponding to decreased electrophoretic mobility. NTg, nontransgenic.

FIGURE 2.

Characterization of αMHC-mTOR^kd and αMHC-mTOR^kd transgenic mice. A, left panel, comparison of the gross morphology and histology of αMHC-mTOR^kd and αMHC-mTOR^ca transgenic and littermate nontransgenic (NTg) control heart. There was no obvious morphological defect in both αMHC-mTOR^kd and αMHC-mTOR^ca transgenic hearts when compared with littermate controls. Right panel, quantitative comparison of the ratio of heart weight versus body weight of αMHC-mTOR^kd and αMHC-mTOR^ca transgenic hearts and nontransgenic littermate control. B, echocardiograph analysis of αMHC-mTOR^kd and αMHC-mTOR^ca transgenic mice and littermate controls (3-month-old male). Representative M-Mode images are shown in panels a–c. Measurements of various parameters and statistics analysis are summarized in panel d. C, ECG recording of the αMHC-mTOR^kd and littermate control mice (3-month-old male). There was a difference in the function of atria and sinoatrial node in αMHC-mTOR^kd hearts compared with littermate controls.

FIGURE 3.

Testing cardiac response of αMHC-mTOR^kd transgenic mice to hypertrophic stimulation. A, comparison ofαMHC-mTOR^kd transgenic and littermate control hearts (3-month-old male) in response to isoproterenol and swimming stimulation. A, panel a, gross morphology of the hearts; panel b, statistic comparison of the ratio of heart weight versus body weight without and with swimming exercise (swim) or isoproterenol treatment (iso). There is no significant difference in change of heart weight after swimming exercise or isoproterenol treatment among these mice. Panel c, morphological comparison of the size of dispersed cardiomyocytes of nontreated control, swimming exercised, and isoproterenol-treated mice. Panel d, statistic comparison of the cell size. Similar degree of hypertrophic response was observed in nontransgenic (NTg) and αMHC-mTOR^kd transgenic cardiomyocytes. B, Western blot analysis of activated Akt level in basal (nonexercised) and exercised hearts. Akt activation is not altered in αMHC-mTOR^kd mice. C, control.

FIGURE 4.

Testing cardiac response of αMHC-mTOR^kd mice to nutrient energy supply and insulin stimulation. A, Western blot analysis of αMHC-mTOR^kd and littermate nontransgenic control mice subjected to fast-refeeding protocol. p70 S6k (Thr-389) was activated by refeeding in control heart, but severely inhibited in αMHC-mTOR^kd heart, whereas Akt activation is not affected in αMHC-mTOR^kd mice. B, Western blot analysis of αMHC-mTOR^kd and littermate nontransgenic control (C) mice subjected to insulin (Ins) stimulation. The levels of phosphorylation of p70 S6k and 4E-BP1 were significantly reduced in αMHC-mTOR^kd hearts when compared with littermate nontransgenic (NTg) control subjected with insulin administration. The skeletal muscle of αMHC-mTOR^kd mice was not affected when compared with controls.

FIGURE 5.

Biochemical analysis of the level of 4E-BP1 bound to eIF4E in αMHC-mTOR^kd mice. A, in nontransgenic (NTg) mice, insulin (Ins) was able to reduce the amount of 4E-BP1 bound to eIF4E. In contrast, the amount of 4E-BP1 bound to eIF4E was not found reduced in αMHC-mTOR^kd heart. B, skeletal muscle sample from identical mice presented in A was used as internal control. C, quantitative and statistic comparison from independent experiments. Each bar represents the average of three experiments, and values are expressed as percentage versus control (set at 100%).