Inhibition of eNOS Partially Blunts the Beneficial Effects of Nebivolol on Angiotensin II-Induced Signaling in H9c2 Cardiomyoblasts

Figures

• 

  Figure 1. Representative DCF fluorescence images and analysis of ROS production in H9c2 cells pretreated with L-NAME (L-N) (100 µM) or L-N plus nebivolol (Neb) (1 µM) prior to Ang II stimulation (A,B). Ang II (1 µM) increased the DCFDA fluorescence, which was not affected by either L-N or L-N plus Neb (C,D). The quantification of fluorescence changes in DCFDA-loaded cells using a fluorescence reader at an excitation and emission of 488 and 522 nm, respectively. Ang II-stimulated an increase in NADP+/NADPH ratio, and NOX2 mRNA expression levels were not reduced by pretreatment with L-N plus Neb (E,F). * p < 0.05 vs. untreated (Con) and NS, nonsignificant vs. Ang II. Values are means ± SEM N ≥ 6 for each treatment group.

• 

  Figure 2. Effects of Neb plus L-N combination on Ang II-induced mRNA expression of cardiac hypertrophy genes in H9c2 cardiomyoblasts. Downregulation of mRNA expression of atrial natriuretic peptide (ANP) (A), brain natriuretic peptide (BNP) (B), and β-myosin heavy chain (β-MHC) (C) by Neb + L-N. (D) Images representing morphological changes in H9c2 cells stained with crystal violet; scale bar = 20 μm. (E) Cell surface area was analyzed and is presented as mean (n = 100 cells/group). * p < 0.05 vs. untreated (Con) and # p < 0.05 vs. Ang II. Values are means ± SEM. N ≥ 3–6 for each treatment group.

• 

  Figure 3. Effects of Neb plus L-N combination on Ang II-induced mTOR signaling in H9c2 cardiomyoblasts. Top: Representative images of autoradiograms showing elevated levels of pSer2448 mTOR (A), pThr389 S6K1 (B), and pSer235/236RPS6 (C) in H9c2 cardiomyoblasts stimulated with Ang II and suppression by Neb and Neb + L-N. Bottom: Graphs show results of densitometry analysis of the intensity of the phosphorylated protein bands after adjusting for the intensity of total protein bands (tmTOR, tS6K1, tRPS6). Top: Representative images of autoradiograms showing elevated levels of pSer2448 mTOR (D), pThr389 S6K1 (E), and pSer235/236RPS6 (F) in H9c2 cardiomyoblasts stimulated with Ang II and suppression by Rap. Bottom: Graphs show results of densitometry analysis of the intensity of the phosphorylated protein bands after adjusting for the intensity of total protein bands (tmTOR, tS6K1, tRPS6). * p < 0.05 vs. untreated (Con) and # p < 0.05 vs. Ang II. Values are means ± SEM. N ≥ 3–6 for each treatment group.

• 

  Figure 4. Effects of Neb plus L-N combination on Ang II-induced mRNA expression of proinflammatory cytokines in H9c2 cardiomyoblasts. mRNA levels of TNF-α (A) and iNOS (B) were not reduced by Neb + L-N. * p < 0.05 vs. untreated (Con) and NS, nonsignificant vs. Ang II. Values are means ± SEM. N ≥ 3–6 for each treatment group.

• 

  Figure 5. Effects of Neb plus compound C (CC) on Ang II-induced ROS generation and mRNA expressions of hypertrophy and inflammatory genes. Ang II-induced increase in DCFDA fluorescence was not affected by pretreatment with CC (5 µM) plus Neb (1 µM) but was significantly reduced by Neb alone (A,B) Ang II-mediated mRNA expression levels of proinflammatory cytokines TNF-α (C) and iNOS (D) were not reduced by CC plus Neb pretreatment. Ang II-mediated mRNA expressions of hypertrophy markers ANP (E), BNP (F), and β-MHC (G) were significantly reduced by CC plus Neb pretreatment. ** p < 0.02, * p < 0.05 vs. untreated (Con). ## p < 0.02, # p < 0.05 vs. Ang II and NS, nonsignificant vs. Ang II. Values are means ± SEM N ≥ 6 for each treatment group.

• 

  Figure 6. Schematic diagram showing that the protective effects of nebivolol on Ang II-induced signaling are partially blunted by inhibition of eNOS and AMPK. NO production via the agonistic activation of β3-AR/AMPK/eNOS pathway by nebivolol is required for its inhibitory effects on Ang II-induced ROS generation and activation of proinflammatory cytokines, such as TNF-α and iNOS. On the contrary, inhibitory effects of nebivolol on Ang II-mediated mTORC1 signaling and hypertrophic response are completely independent of NO bioavailability.