Figure 2: The H
2019-06-11T07:09:15Z (GMT) by
(A and C) Cardiomyocytes were pretreated for 1 h with 10 μM GF109203X (GFX), 10 μM PP1, 10 μM H89, 10 μM Gö6976, 0.1 μM propranolol (prop), 1 μM isoproterenol (Iso), 1 μM carvedilol (carv), 10 μM pindolol (pin), 10 μM timolol (tim), 10 μM atenolol (aten), or 10 μM metoprolol (met) as indicated and then challenged with vehicle or 0.1 mM H2O2 for 60 min (unless indicated otherwise). (B) Treatment with vehicle or the indicated concentrations H2O2 (in the absence or presence of 1 μM carvedilol) followed a 24-h pre-incubation with 100 ng/ml pertussis toxin (PTX). Experiments in A to C were performed in parallel on cardiomyocyte cultures that did or did not overexpress the β1AR transgene to compare stimulus-induced changes in native rat β1ARs (tracked with SC anti-β1AR antibody) and heterologously overexpressed human β1ARs (tracked with SC and/or Abcam anti-β1AR antibodies as indicated). Because β-actin immunoreactivity was not altered by β1AR overexpression, a single β-actin blot from uninfected cultures is depicted in the figures as a protein loading control. H2O2 and carvedilol-dependent changes in endogenous or heterologously overexpressed β1AR immunoreactivity are quantified in A, right (n = 6). (D) β1AR-overexpressing cardiomyocytes were treated for 24 h with a panel of β1AR ligands (at concentrations stipulated in A). Effects on β1AR transgene abundance are depicted on top, with results for 3 separate experiments on different culture preparations quantified on the bottom (*p < 0.05 by analysis of variance followed by a Tukey post hoc analysis). For quantification of immunoreactivity (which is expressed as arbitrary units), levels of the truncated β1AR species in ligand-treated cultures (gray bars) were normalized to the level of the truncated β1AR species in the corresponding vehicle-treated culture (black bar), which was set to 100%. (E) β1AR-overexpressing cardiomyocytes were pretreated for 24 h with vehicle, 1 μM carvedilol, or 5 μM mitoTempo (mitoT, Sigma-Aldrich, St. Louis, Missouri) and then challenged with 100 μM H2O2 as indicated. Lysates were probed for β1AR immunoreactivity with β-actin immunoreactivity included as a loading control. The experiment is representative of data obtained in 4 separate experiments on different culture preparations. (F) Lysates from cardiomyocytes treated for 1 h with vehicle or 100 μM H2O2 (following a 1-h pretreatment with vehicle or 1 μM carvedilol as indicated) were probed for cyclic adenosine monophosphate binding response element–protein (CREB) phosphorylation and CREB protein expression. All immunoblotting data represent results obtained in 3 to 5 separate experiments. Abbreviations as in Figure 1.