Exercise training improves cardiac function and exercise tolerance in myocardial infarction-induced heart failure.

<p>Schematic panel (A). Fractional shortening (B), distance run (C), heart rate (D) and blood pressure (E) in control (sham, white bars), MI-HF (gray bars) and MI-HF exercise trained (MI-HFtr, gray bars) rats before and after 8 wks of either sedentary or exercise training protocol. Error bars indicate SEM. Interaction between main effects: fractional shortening [F (2, 15) = 5.28, p = 0.0183]; distance run [F (2, 31) = 48.97, p<0.0001]; heart rate [F (2, 13) = 4.06, p = 0.0425] and blood pressure [F (2, 13) = 1.05, p = 0.3764]. §, p<0.05 vs. before experimental protocol. *, p<0.05 vs. control (sham) rats. ‡, p<0.05 vs. MI-HFtr rats.</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

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

Echocardiographic measurements.

<p>Left ventricular ejection fraction (EF), interventricular septum in diastole (IVSd), interventricular septum in systole (IVSs), left ventricular end-diastolic diameter (LVEdD), left ventricular end-systolic diameter (LVEsD), left ventricular posterior wall in diastole (LVPWd) and left ventricular posterior wall in systole (LVPWs) were obtained before and after 8 wks of the experimental protocol in control (sham), MI-HF and MI-HF exercise trained (MI-HFtr) rats (Mean ± SEM). Interaction between main effects: EF [F (2, 15) = 6.84, p = 0.0077] *control>MI-HF (p<0.0001) and MI-HFtr (p<0.0001) before and after experimental protocol, ‡MI-HFtr>MIHF after experimental protocol (p = 0.0136) and §MI-HFtr beforeafter experimental protocol (p = 0.0401).</p>€<p>Main time effect: IVSs [F (1, 15) = 5.98, p = 0.0272] pre-training values</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

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

Exercise training improves oxygen consumption and reduces H₂O₂ release in cardiac isolated mitochondria from myocardial infarction-induced heart failure animal.

<p>Mitochondrial state 3 (A) and state 4 (B) respiratory rates; respiratory control ratio (C); maximum calcium uptake (D) and H₂O₂ release (E) 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. All measurements were performed in the ventricular remote area. Error bars indicate SEM. Mitochondrial state 3 [F (2, 41) = 8.62, p = 0.0007] and state 4 [F (2, 41) = 8.86, p = 0.0006] respiratory rates; respiratory control ratio [F (2, 45) = 3.26, p = 0.0475]; maximum calcium uptake [F (2, 14) = 5.72, p = 0.0152] and H₂O₂ release [F (2, 37) = 5.28, p = 0.0095]. *, p<0.05 vs. control (sham) rats. ‡, p<0.05 vs. MI-HFtr rats.</p

Exercise reestablishes autophagic flux and mitochondrial quality control in heart failure

<p>We previously reported that facilitating the clearance of damaged mitochondria through macroautophagy/autophagy protects against acute myocardial infarction. Here we characterize the impact of exercise, a safe strategy against cardiovascular disease, on cardiac autophagy and its contribution to mitochondrial quality control, bioenergetics and oxidative damage in a post-myocardial infarction-induced heart failure animal model. We found that failing hearts displayed reduced autophagic flux depicted by accumulation of autophagy-related markers and loss of responsiveness to chloroquine treatment at 4 and 12 wk after myocardial infarction. These changes were accompanied by accumulation of fragmented mitochondria with reduced O₂ consumption, elevated H₂O₂ release and increased Ca²⁺-induced mitochondrial permeability transition pore opening. Of interest, disruption of autophagic flux was sufficient to decrease cardiac mitochondrial function in sham-treated animals and increase cardiomyocyte toxicity upon mitochondrial stress. Importantly, 8 wk of exercise training, starting 4 wk after myocardial infarction at a time when autophagy and mitochondrial oxidative capacity were already impaired, improved cardiac autophagic flux. These changes were followed by reduced mitochondrial number:size ratio, increased mitochondrial bioenergetics and better cardiac function. Moreover, exercise training increased cardiac mitochondrial number, size and oxidative capacity without affecting autophagic flux in sham-treated animals. Further supporting an autophagy mechanism for exercise-induced improvements of mitochondrial bioenergetics in heart failure, acute in vivo inhibition of autophagic flux was sufficient to mitigate the increased mitochondrial oxidative capacity triggered by exercise in failing hearts. Collectively, our findings uncover the potential contribution of exercise in restoring cardiac autophagy flux in heart failure, which is associated with better mitochondrial quality control, bioenergetics and cardiac function.</p