mTOR Complexes Repress Hypertrophic Agonist–Stimulated Expression of Connective Tissue Growth Factor in Adult Cardiac Muscle Cells

Abstract

Connective tissue growth factor (CTGF) is a fibrogenic cytokine that promotes fibrosis in various organs. In the heart, both cardiomyocytes (CM) and cardiac fibroblasts have been reported as a source of CTGF expression, aiding cardiac fibrosis. Although the mammalian target of rapamycin (mTOR) forms 2 distinct complexes, mTORC1 and mTORC2, and plays a central role in integrating biochemical signals for protein synthesis and cellular homeostasis, we explored its role in CTGF expression in adult feline CM. CM were stimulated with 10 μM phenylephrine (PE), 200 nM angiotensin (Ang), or 100 nM insulin for 24 hours. PE and Ang, but not insulin, caused an increase in CTGF mRNA expression with the highest expression observed with PE. Inhibition of mTOR with torin1 but not rapamycin significantly enhanced PE-stimulated CTGF expression. Furthermore, silencing of raptor and rictor using shRNA adenoviral vectors to suppress mTORC1 and mTORC2, respectively, or blocking phosphatidylinositol 3-kinase (PI3K) signaling with LY294002 (LY) or Akt signaling by dominant-negative Akt expression caused a substantial increase in PE-stimulated CTGF expression as measured by both mRNA and secreted protein levels. However, studies with dominant-negative delta isoform of protein kinase C demonstrate that delta isoform of protein kinase C is required for both agonist-induced CTGF expression and mTORC2/Akt-mediated CTGF suppression. Finally, PE-stimulated CTGF expression was accompanied with a corresponding increase in Smad3 phosphorylation and pretreatment of cells with SIS3, a Smad3 specific inhibitor, partially blocked the PE-stimulated CTGF expression. Therefore, a PI3K/mTOR/Akt axis plays a suppressive role on agonist-stimulated CTGF expression where the loss of this mechanism could be a contributing factor for the onset of cardiac fibrosis in the hypertrophying myocardium.

Connective tissue growth factor dependent collagen gene expression induced by MAS agonist AR234960 in human cardiac fibroblasts

“…In experimental rat models, AngII-induction of CTGF mRNA expression was found to lead to cardiac fibroblast activation during chronic HF [ 25 ]. Studies in feline isolated adult cardiomyocytes indicate that PE and AngII induced CTGF expression and release during maladaptive remodeling [ 26 ]. However, genetic overexpression of CTGF in rat cardiomyocytes was shown to promote cardiac hypertrophy but not fibrosis and protect against pressure overload [ 27 ].…”

Reversal of maladaptive fibrosis and compromised ventricular function in the pressure overloaded heart by a caveolin-1 surrogate peptide

“…GAPDH was used as an internal control for normalizing target gene expression levels as described. 40 …”

Poly(ADP‐ribose) polymerase 1 induces cardiac fibrosis by mediating mammalian target of rapamycin activity

“…CFs were incubated with 3AB (10 μM, 24 hours) or ABT-888 (10 μM, 24 hours) followed by incubation with TGF-β1 (10 ng/mL for 24 hours). [11][12][13][14][15][16][17][18][19][20][21] It is mainly due to the effect of mTOR on collagen expression, Smad3, p53/JNK-mediated F I G U R E 6 Involvement of mTOR in PARP1-mediated cardiac fibrosis in vitro. B and G, The protein levels of PARP1 and mTOR were determined by Western blot analysis.…”

“…8 In particular, activated mTORC1 positively regulates protein synthesis by phosphorylating its downstream substrates, such as p70 ribosomal S6 kinase 1 (S6K1), eukaryotic initiation factor 4E-binding protein 1 (4EBP, eIF4Ebinding protein 1), and UNC51like kinase 1 (ULK1). [11][12][13][14][15][16][17][18][19][20][21] It is mainly due to the effect of mTOR on cardiac collagen expression, Smad3, p53/JNK-mediated apoptosis, autophagy, and inflammatory response. [11][12][13][14][15][16][17][18][19][20][21] It is mainly due to the effect of mTOR on cardiac collagen expression, Smad3, p53/JNK-mediated apoptosis, autophagy, and inflammatory response.…”

“…10 It is well documented that mTOR signaling is associated with many cardiac diseases accompanied by cardiac fibrosis, including cardiac hypertrophy, dysfunction, dilated cardiomyopathy, and ischemia-reperfusion injury. [11][12][13][14][15][16][17][18][19][20][21] It is mainly due to the effect of mTOR on cardiac collagen expression, Smad3, p53/JNK-mediated apoptosis, autophagy, and inflammatory response. 12,17,19,21 Poly(ADP-ribose) polymerase 1 (PARP1), the founding subtype of the PARPs enzyme family, attaches the polymers of ADP-ribose (PAR) to target proteins and consumes the cellular NAD pool.…”