Abstract P623: Nutrient Stress Response of Cardiovascular Cells to β-Blockers, ARB and AT2R Agonists
To determine how cardioprotective drugs modulate cell response to nutrient starvation, we investigated the effects of β-blockers (Nebivolol [Neb], Carvedilol [Car], Metoprolol [Met] and Atenolol [Aten] (3μM each), Angiotensin II (Ang II) (300nM), AT1R blocker (ARB-Losartan (1μM)) and AT2R agonists (CGP42112A [CGP] and a novel peptide agonist NP-6A4 (300nM each)) on serum-starved HL-1 cardiomyocytes. The Xcelligence Real-Time Cell Analyzer (RTCA), which measures area covered by cells in microtiter plates and displays it as a unitless quantity called Cell Index (CI), was used to assess cellular changes in response to drug treatment. To find whether changes in CI are due to altered proliferation or cell size, we utilized the MTS Proliferation Assay, a colorimetric assay that measures formazan dye produced by viable cells, and fluorophore-conjugated Wheat Germ Agglutinin (WGA) labeling to measure cell size respectively. Difference in CI units is reported as % of CI units of cells treated with vehicle. CI units were suppressed by β-blockers (Aten≤15%; Met≤15%; Neb≤17%; Car≤8%), increased by Ang II (≥9.6%), CGP (≥14%) and NP-6A4 (≥25%), but not by losartan (n≥4 and p≤0.05 for all treatments). MTS assay revealed that only NP-6A4 increased (19%) formazan dye as measured at 490nm (n=3, p<0.05). Differences in cell size of WGA stained cells was calculated as % of size of cells treated with vehicle. Neb and Car decreased cell size (Neb≤14%, Car≤10%, p<0.05 and p<0.1 respectively), suggesting their mechanism for decrease in CI. Other treatments showed no significant change. Next, we determined changes in Myeloid cell leukemia 1 (MCL-1) levels, an essential protein for cardiomyocyte survival, in response to drug treatments by immunoblotting and immunofluorescence. β-Blockers suppressed MCL-1 expression in HL-1 cardiomyocytes (Neb≤26%, Car≤24%, Met≤24%, Aten≤16%) while AT2R agonists increased (CGP≥17%, NP-6A4≥28%, n=3; p<0.05 for all). Treatment of Female Human Coronary Artery Vascular Smooth Muscle Cells by these drugs also showed a similar expression pattern of expression for MCL-1 (CGP≥23%, NP-6A4≥43%). Therefore, AT2R agonist NP-6A4 is more effective in protecting serum starved cardiovascular cells.
Author Disclosures: A. Mahmood: None. L. Pulakat: None.
- © 2015 by American Heart Association, Inc.