Limits...
Adiponectin modulates oxidative stress-induced autophagy in cardiomyocytes.

Essick EE, Wilson RM, Pimentel DR, Shimano M, Baid S, Ouchi N, Sam F - PLoS ONE (2013)

Bottom Line: However, neither H2O2 nor APN affected ATG5, ATG7, or Beclin-1 expression.H2O2 decreased phospho-mTOR by 36±13%, which was restored by APN.These findings demonstrate the anti-oxidant potential of APN in oxidative stress-associated cardiovascular diseases, such as hypertension-induced HF-preserved EF.

View Article: PubMed Central - PubMed

Affiliation: Whitaker Cardiovascular Institute, Boston University School of Medicine, Boston, Massachusetts, United States of America.

ABSTRACT
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 (30 µg/mL) followed by 1mM hydrogen peroxide (H2O2) exposure. Wild type (WT) and APN-deficient (APN-KO) mice were infused with angiotensin (Ang)-II (3.2 mg/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.

Show MeSH

Related in: MedlinePlus

APN-attenuates H2O2-mediated autophagy in ARVM.(A) 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) Representative Western blot. (C) 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). (D) Treatment with H2O2 (iii) induced the formation of the autophagosome as indicated by green puncta marking the cell perimeter vs. control (i). Pretreatment with APN (ii) led to a reduced number of H2O2-induced punctate autophagosomes (iv).
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3716763&req=5

pone-0068697-g002: APN-attenuates H2O2-mediated autophagy in ARVM.(A) 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) Representative Western blot. (C) 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). (D) Treatment with H2O2 (iii) induced the formation of the autophagosome as indicated by green puncta marking the cell perimeter vs. control (i). Pretreatment with APN (ii) led to a reduced number of H2O2-induced punctate autophagosomes (iv).

Mentions: Autophagosome formation is indicative of autophagic activity. Microtubule-associated protein light chain 3 (LC3) is involved in autophagy and exists in two forms: LC3-I is the free cytosolic form, while LC3-II is conjugated to phosphotidylethanolamine (PE) and is incorporated in the autophagosome membrane [38]. LC3-II and LC3-I protein expression were measured by Western blot and the ratio of LC3-II to LC3-I protein expression was used as a measurement of autophagosome formation [39] and as an indirect indication of autophagy. In ARVM, 1mM H2O2 increased the LC3-II/LC3-I ratio by a factor of 3.4±1.0 (p<0.05 vs. control; Figure 2A). Pretreatment with APN abrogated this increased LC3-II/LC3-I ratio (p<0.05 vs. H2O2-treated cells; Figure 2A-B). To corroborate these findings, green fluorescent protein (GFP)-labeled LC3 (GFP-LC3) (10moi) expressing ARVMs were treated with 1mM H2O2 in the presence or absence of APN and analyzed under 60x oil magnification (Figure 2C-D). H2O2 increased the number of GFP-LC3 puncta per cell by a factor of 2.7±0.2 (p<0.001 vs. control), and pretreatment with APN attenuated this increase (p<0.01 vs. H2O2-treated cells). Although increased LC3-II/LC3-I ratio suggests autophagosome accumulation, increased p62 expression suggests defects in the lysosomal end of the pathway, we thus measured p62 expression in H2O2 stimulated ARVM. H2O2 (1mM) increased p62 expression (p<0.01 vs. control) and pretreatment with APN attenuated this increase (p<0.01 vs. H2O2-treated cells; Figure S1 A-B).


Adiponectin modulates oxidative stress-induced autophagy in cardiomyocytes.

Essick EE, Wilson RM, Pimentel DR, Shimano M, Baid S, Ouchi N, Sam F - PLoS ONE (2013)

APN-attenuates H2O2-mediated autophagy in ARVM.(A) 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) Representative Western blot. (C) 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). (D) Treatment with H2O2 (iii) induced the formation of the autophagosome as indicated by green puncta marking the cell perimeter vs. control (i). Pretreatment with APN (ii) led to a reduced number of H2O2-induced punctate autophagosomes (iv).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3716763&req=5

pone-0068697-g002: APN-attenuates H2O2-mediated autophagy in ARVM.(A) 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) Representative Western blot. (C) 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). (D) Treatment with H2O2 (iii) induced the formation of the autophagosome as indicated by green puncta marking the cell perimeter vs. control (i). Pretreatment with APN (ii) led to a reduced number of H2O2-induced punctate autophagosomes (iv).
Mentions: Autophagosome formation is indicative of autophagic activity. Microtubule-associated protein light chain 3 (LC3) is involved in autophagy and exists in two forms: LC3-I is the free cytosolic form, while LC3-II is conjugated to phosphotidylethanolamine (PE) and is incorporated in the autophagosome membrane [38]. LC3-II and LC3-I protein expression were measured by Western blot and the ratio of LC3-II to LC3-I protein expression was used as a measurement of autophagosome formation [39] and as an indirect indication of autophagy. In ARVM, 1mM H2O2 increased the LC3-II/LC3-I ratio by a factor of 3.4±1.0 (p<0.05 vs. control; Figure 2A). Pretreatment with APN abrogated this increased LC3-II/LC3-I ratio (p<0.05 vs. H2O2-treated cells; Figure 2A-B). To corroborate these findings, green fluorescent protein (GFP)-labeled LC3 (GFP-LC3) (10moi) expressing ARVMs were treated with 1mM H2O2 in the presence or absence of APN and analyzed under 60x oil magnification (Figure 2C-D). H2O2 increased the number of GFP-LC3 puncta per cell by a factor of 2.7±0.2 (p<0.001 vs. control), and pretreatment with APN attenuated this increase (p<0.01 vs. H2O2-treated cells). Although increased LC3-II/LC3-I ratio suggests autophagosome accumulation, increased p62 expression suggests defects in the lysosomal end of the pathway, we thus measured p62 expression in H2O2 stimulated ARVM. H2O2 (1mM) increased p62 expression (p<0.01 vs. control) and pretreatment with APN attenuated this increase (p<0.01 vs. H2O2-treated cells; Figure S1 A-B).

Bottom Line: However, neither H2O2 nor APN affected ATG5, ATG7, or Beclin-1 expression.H2O2 decreased phospho-mTOR by 36±13%, which was restored by APN.These findings demonstrate the anti-oxidant potential of APN in oxidative stress-associated cardiovascular diseases, such as hypertension-induced HF-preserved EF.

View Article: PubMed Central - PubMed

Affiliation: Whitaker Cardiovascular Institute, Boston University School of Medicine, Boston, Massachusetts, United States of America.

ABSTRACT
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 (30 µg/mL) followed by 1mM hydrogen peroxide (H2O2) exposure. Wild type (WT) and APN-deficient (APN-KO) mice were infused with angiotensin (Ang)-II (3.2 mg/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.

Show MeSH
Related in: MedlinePlus