Adiponectin Modulates Oxidative Stress-Induced Autophagy in Cardiomyocytes

<div><p>Diastolic heart failure (HF) i.e., “HF with preserved ejection fraction” (HF-preserved EF) accounts for up to 50% of all HF presentations; however there have been no therapeutic advances. This stems in part from an incomplete understanding about HF-preserved EF. Hypertension is the major cause of HF-preserved EF whilst HF-preserved EF is also highly associated with obesity. Similarly, excessive reactive oxygen species (ROS), i.e., oxidative stress occurs in hypertension and obesity, sensitizing the heart to the renin-angiotensin-aldosterone system, inducing autophagic type-II programmed cell death and accelerating the propensity to adverse cardiac remodeling, diastolic dysfunction and HF. Adiponectin (APN), an adipokine, mediates cardioprotective actions but it is unknown if APN modulates cardiomyocyte autophagy. We tested the hypothesis that APN ameliorates oxidative stress-induced autophagy in cardiomyocytes. Isolated adult rat ventricular myocytes were pretreated with recombinant APN (30µg/mL) followed by 1mM hydrogen peroxide (H₂O₂) exposure. Wild type (WT) and APN-deficient (APN-KO) mice were infused with angiotensin (Ang)-II (3.2mg/kg/d) for 14 days to induced oxidative stress. Autophagy-related proteins, mTOR, AMPK and ERK expression were measured. H₂O₂ induced LC3I to LC3II conversion by a factor of 3.4±1.0 which was abrogated by pre-treatment with APN by 44.5±10%. However, neither H₂O₂ nor APN affected ATG5, ATG7, or Beclin-1 expression. H₂O₂ increased phospho-AMPK by 49±6.0%, whilst pretreatment with APN decreased phospho-AMPK by 26±4%. H₂O₂ decreased phospho-mTOR by 36±13%, which was restored by APN. ERK inhibition demonstrated that the ERK-mTOR pathway is involved in H₂O₂-induced autophagy. Chronic Ang-II infusion significantly increased myocardial LC3II/I protein expression ratio in APN-KO vs. WT mice. These data suggest that excessive ROS caused cardiomyocyte autophagy which was ameliorated by APN by inhibiting an H₂O₂-induced AMPK/mTOR/ERK-dependent mechanism. These findings demonstrate the anti-oxidant potential of APN in oxidative stress-associated cardiovascular diseases, such as hypertension-induced HF-preserved EF.</p> </div

Effect of APN on H₂O₂ mediated increase on phospho-ERK and phospho-AMPK protein expression.

<p>(<b>A</b>) 1mM H₂O₂ (15min) increased phospho-ERK protein expression by a factor of 2.1±0.3 (#p<0.01 vs. control) and was attenuated by pretreatment with APN by 40±7% (*p<0.05 vs. H₂O₂ treated cells). (<b>B</b>) Representative Western blot.</p

APN-attenuates H₂O₂-mediated autophagy in ARVM.

<p>(<b>A</b>) 1mM H₂O₂ increased LC3II/I protein expression ratio in ARVM by a factor of 3.4±1.0 (*p<0.05 vs. control). This was abrogated by pretreatment with APN (58±10% reduction; *p<0.05 vs. H₂O₂-treated cells). (<b>B</b>) Representative Western blot. (<b>C</b>) ARVMs were transfected with GFP-labeled LC3 virus (10moi) to visualize the presence of autophagosomes. 1mM H₂O₂ increased the number of GFP-LC3 puncta per cell by a factor of 2.7±0.2 vs. control; (‡p<0.001). Pretreatment with APN decreased this by 45±3% vs. H₂O₂-treated cells (**p<0.01). (<b>D</b>) Treatment with H₂O₂ (iii) induced the formation of the autophagosome as indicated by green puncta marking the cell perimeter vs. control (<b>i</b>). Pretreatment with APN (ii) led to a reduced number of H₂O₂-induced punctate autophagosomes (<b>iv</b>).</p

Loss of APN enhances autophagy in response to Ang-II infusion in vivo.

<p>WT and APN-KO mice were infused with Ang-II (3.2mg/kg/d) for 14 days, and LC3 gene and LC3II/I protein expression ratio was assessed from the LV of the hearts. (<b>A</b>) LC3 gene expression was increased in 37±1.1% in Ang-II infused APN-KO mice vs. WT Ang-II infused mice (*p<0.05) (<b>B</b>) LC3II/I protein expression was increased in Ang-II infused APN-KO mice by a factor of 2.7±0.6 vs. WT Ang-II infused mice (*p<0.05). (<b>B</b>) Representative Western blot.</p

H₂O₂ decreased cell viability.

<p>(<b>A</b>) Myocytes were exposed to various concentrations of H₂O₂ for 90 min and viability was assessed by MTT uptake. Data are the mean from three experiments. ARVM were exposed to H₂O₂ in concentrations ranging from 10 to 1000 µM H₂O₂. (<b>B</b>) Adult cardiomyocytes treated with 1mM H₂O₂ resulted in a 34±2% decrease in viability (‡p<0.001 vs. control) as measured by MTT assay. APN pretreatment (30µg/mL), before H₂O₂ exposure, increased cell viability by 19±4% (**p<0.01 vs. H₂O₂-treated).</p

Effect on upstream regulators of autophagy.

<p>(<b>A</b>) 1mM H₂O₂ (15min) diminished phospho-mTOR expression by 36±13% (*p<0.05 vs. control). Pretreatment with APN restored phospho-mTOR protein expression to baseline levels (*p<0.05 vs. H₂O₂ treated cells). (<b>B</b>) Representative Western blot of mTOR expression. (<b>C</b>) 1mM H₂O₂ (15min) decreased beclin-1 protein expression by 35±2% (‡p<0.001 vs. control). Pretreatment with APN had no significant effect on beclin-1 protein expression. (<b>D</b>) Representative Western blot of beclin-1 protein expression.</p

H₂O₂ stimulation involves AMPK/mTOR pathway.

<p>(<b>A</b>) 1mM H₂O₂ (15min) increased phospho-AMPK protein expression in ARVMs by 56±4% (#p<0.001 vs. control) and APN pretreatment decreased H₂O₂-induced phospho-mTOR expression by 28±3% (#p<0.001 vs. H₂O₂). (<b>B</b>) Representative Western blot of p-AMPK expression.</p

Correlation with dROMs levels.

<p>Correlation with dROMs levels.</p

Correlation with Log hsCRP levels.

<p>Correlation with Log hsCRP levels.</p

Correlation with Log Fstl1 levels.

<p>Correlation with Log Fstl1 levels.</p