Aortic β-AR subtypes expression.

<p>Protein expression of β₁- β₂- and β₃-adrenoceptors (AR) evaluated in the membrane fraction of aortas from WT, β₁KO and β₂KO mice treated for 7 days with vehicle or isoproterenol (ISO). (A) Representative Western-blot autoradiographs for each β-AR subtype in membrane preparations of aorta and positive controls (+C: heart for β₁-AR; skeletal muscle for β₂-AR; adipose tissue for β₃-AR). Densitometric quantification was evaluated for β₁- (B) and β₂-AR (C) but not for β₃-AR, as this subtype was not expressed (n.e.) in the mouse aorta. The number of samples analyzed (pool of 3 aortas in each sample) is indicated in the bar for each group. Values (mean ± SEM) are expressed the fold-change in β-AR expression compared to the WT. Significance was assessed using a 2-way ANOVA.</p

Isoproterenol treatment induces β₂-AR-Gi-ERK1/2 pathway activation and eNOS uncoupling.

<p>Protein expression of Giα-1,2 (A), Giα-3 (B), ERK 1/2 phosphorylated at Thr²⁰² and Tyr²⁰⁴ (C), p38 MAPK phosphorylated at Thr¹⁸⁰ and Tyr¹⁸² (D) and eNOS protein dimerization (E) in aortas from control and 7-day isoproterenol-treated (ISO) wild-type (WT) and β₂KO mice. The top panels in each graph represent typical Western-blot autoradiographs. Giα protein expression was normalized to the α-actin content in each sample, and phosphorylated ERK 1/2 and p38 MAPK were normalized to the total content of ERK 1/2 and p38 MAPK, respectively, and these results were expressed as the fold-change compared to WT aorta. eNOS dimerization was expressed as ratio of dimer:monomer band intensity. The number of animals used in each group is indicated in the bars. Values are presented as the mean ± SEM. Significance was assessed using a 2-way ANOVA: *p<0.05 <i>vs.</i> WT.</p

Inhibition of Giα protein or ERK1/2 activation reversed hypercontractility to phenylephrine induced by β-AR overactivation in aorta of wild-type, but not in β₂KO mice.

<p>Effect of pertussis toxin (PTx, 4 μM) and PD98,059 (1 μM) on the concentration-response curves to phenylephrine in aortic rings of wild-type (WT) (A, D) and β₂KO (B, E) mice treated for 7 days with vehicle or isoproterenol (ISO). The contraction response is expressed as a % of the contraction to KCl (125 mM). Bar graphs show differences in the area under the concentration-response curve (AUC) in the presence or absence of PTx (C) or PD98,059 (F) in WT and β₂KO mice treated or not with ISO. Values are presented as the mean ± SEM. The number of animals used in each group is indicated in parenthesis. Significance was assessed using a 2-way ANOVA: ⁺p<0.05 <i>vs.</i> WT ISO; *p<0.05 <i>vs.</i> WT.</p

Role of superoxide anion and NOS in the vascular effect of isoproterenol treatment.

<p>Effect of L-NAME (LN, 100 μM) or superoxide dismutase (SOD, 150 U/mL) on the concentration-response curves to phenylephrine of vehicle- (open symbols) or 7-day isoproterenol-treated (ISO, close symbols) aortic rings from wild-type (A, D), β₁KO (B, E) and β₂KO (C, F) mice. The contraction response is expressed as a % of the contraction to KCl (125 mM). Values are presented at the mean ± SEM. E+ =  intact endothelium. The number of animals used in each group is indicated in parenthesis. Significance was assessed using a 2-way ANOVA: *p<0.05, **p<0.01 <i>vs</i>. WT E+; ⁺p<0.05, ⁺⁺p<0.01 <i>vs.</i> β₁KO E+; ^#p<0.05 <i>vs</i>. β₂KO E+.</p

Giα protein activity mediates the vascular oxidative stress induced by isoproterenol.

<p>Panel A shows representative fluorographs of microscopic sections of thoracic aorta from wild-type (WT) and β₂KO mice treated for 7 days with vehicle or isoproterenol (ISO). Vessels were labeled with the oxidative dye hydroethidine, which produces a red fluorescence when oxidized to ethidium bromide. Panel B shows the densitometric analysis of the ethidium-bromide-positive nuclei evaluated under basal conditions or incubated with apocynin (APO, 30 μM), L-NAME (LN, 100 μM), PTx (4 μM) or superoxide dismutase (SOD, 150 U/mL). The fluorescence signal was evaluated as the intensity of fluorescence per pixel normalized by vessel area. Values are presented as the mean ± SEM. N = 4–7 animals in each group. Significance was assessed using a 2-way ANOVA: *p<0.05 <i>vs</i>. respective basal values for each group; ^#p<0.05 <i>vs</i>. basal WT value; ⁺p<0.05 <i>vs</i>. ISO-treated WT value.</p

Physiological parameters.

<p>Peak VO₂ (in mL O₂·kg⁻¹·min⁻¹), body weight (BW in grams), <i>soleus</i> muscle citrate synthase activity (CS in µmol·mg⁻¹·min⁻¹), heart weight/body weight ratio (HW/BW), myocardial infarction (MI) area, cardiomyocyte width (µm) and cardiac collagen content (%) data in control (sham), MI-HF and MI-HF exercise trained (MI-HFtr) rats (Mean ± SEM).</p>€<p>Main time effect: peak VO₂ [F (1, 18) = 9.75, p = 0.0058] pre-training values>post-training values and BW [F (1, 16) = 10.73, p = 0.0047]. CS activity [F (2, 21) = 29.80, p<0.0001] *MI-HFcontrol (p = 0.0002); HW/BW [F (2, 16) = 8.55, p = 0.0029] *controlcontrol (p<0.0001) and cardiac collagen content [F (2, 23) = 3.76, p = 0.0245] *MI-HF>control (p = 0.0189) and ‡MI-HFtr (p = 0.0311).</p

Exercise training improves protein quality control in myocardial infarction-induced heart failure.

<p>Oxidized protein levels (A), soluble oligomers accumulation (B), HSP25 (C) αβ-crystallin (D) protein levels in heart samples from 24 week-old control (sham, white bars), MI-HF (gray bars) and MI-HF exercise trained (MI-HFtr, gray bars) rats. Representative blots of oxidized protein, soluble oligomers, HSP25, αβ-crystallin and GAPDH (E). All measurements were performed in the ventricular remote area. Protein expression was normalized by GAPDH. Error bars indicate SEM. Oxidized protein levels [F (2, 19) = 5.25, p = 0.0312]; soluble oligomers accumulation [F (2, 15) = 3.97, p = 0.0412]; HSP25 [F (2, 19) = 4.21, p = 0.0306] and αβ-crystallin proteins levels [F (2, 17) = 1.49, p = 0.0252]. *, p<0.05 vs. control (sham) rats. ‡, p<0.05 vs. MI-HFtr rats.</p

Exercise training decreases 4-HNE modification of proteasome and re-establishes cardiac ubiquitin-proteasome system function in myocardial infarction-induced heart failure.

<p>(A) 4-HNE protein adducts in heart samples from 24 week-old control (sham), MI-HF and MI-HF exercise trained (MI-HFtr) rats. Protein expression was normalized by GAPDH. Inset: Representative blot of 4-HNE protein adducts. Black arrows indicate changes in the adduct formation in MI-HF and MI-HFtr samples, red arrows indicate changes in the adduct formation of proteins at the molecular weight of proteasomal subunits. (B) 20S proteasome subunits (α5/α7, β1, β5, β7) were precipitated from left ventricle tissue from 24-week-old control, MI-HF and MI-HFtr rats (B, n = 3 per group), and then probed with 4-HNE-modified proteins antibody. Equal sample loading was verified using α5/α7, β1, β5 and β7 proteasome subunits antibody. (C) Chymotrypsin-like activity of 26S proteasome, (D) 20S proteasome α5/α7, β1, β5, β7 protein levels and (E) polyubiquitinated proteins levels in heart samples from 24 week-old control, MI-HF and MI-HFtr rats. Protein expression was normalized by GAPDH. (F) Representative blots of polyubiquitinated proteins, 20S proteasome and GAPDH. All measurements were performed in the ventricular remote area. Error bars indicate SEM. 4-HNE protein adducts [F (2, 15) = 42.58, p<0.0001]; chymotrypsin-like activity of 26S proteasome [F (2, 25) = 12.90, p = 0.0001]; 20S proteasome α5/α7, β1, β5, β7 [F (2, 18) = 0.81, p = 0.4595] and polyubiquitinated proteins levels [F (2, 18) = 4.19, p = 0.0318]. *, p<0.05 vs. control (sham) rats. ‡, p<0.05 vs. MI-HFtr rats.</p

4-HNE irreversibly inactivates 20S proteasome <i>in vitro</i>.

<p>(A) Schematic panel of <i>in vitro</i> incubations. (B) Purified 20S proteasome (1 ug) was incubated for 30 min at 37°C with 4-HNE (10 or 100 µM) and proteasomal activity was measured at the end of incubation. DTT (1μ) was added to the reaction either previous or after 4-HNE incubations. Of interest, prior, but no later, incubation with DTT protected 4-hydroxi-2-nonenal inhibition of proteasomal activity. Error bars indicate SEM. Proteasomal activity [F (7, 32) = 21.37, p<0.0001]. *, p<0.05 vs. control, 4-HNE (10 µM)+DTT (before). #, p<0.05 vs. 4-HNE (10 µM).</p

Oxidative stress contributes to proteasomal inactivation, accumulation of damaged proteins and cell death in cultured neonatal cardiomyocytes.

<p>Proteasomal activity (A), oxidized protein levels and representative blots (B) and cell death (C) in cultured neonatal cardiomyocytes. Concordance between proteasomal activity, oxidized protein levels and cell death in cultured neonatal cardiomyocytes (D). Cells were stimulated with antimycin A (100 µM, Ant A, gray bars) or H₂O₂ (100 µM, gray bars) or Epoxomicin (1 µM, EPO, gray bars) for 2 hours. Measurements were performed 24 hrs after treatments. Experiments were repeated at least 5 times. Protein expression was normalized by GAPDH. Error bars indicate SEM. Proteasomal activity [F (3, 20) = 30.85, p<0.0001]; oxidized protein levels [F (2, 9) = 21.84, p = 0.0003] and cell death [F (3, 14) = 27.53, p<0.0001]. #, p<0.05 vs. non-treated cells (control). $, p<0.05 vs. antimycin A- and Epoxomicin-treated cells.</p