J Vet Sci.  2017 Jun;18(2):129-140. 10.4142/jvs.2017.18.2.129.

Apoptosis in response to heat stress is positively associated with heat-shock protein 90 expression in chicken myocardial cells in vitro

• Zhang XH
• Wu H
• Tang S
• Li QN
• Xu J
• Zhang M
• Su YN
• Yin B
• Zhao QL
• Kemper N
• Hartung J
• Bao ED

• KMID: 2412565
• DOI: http://doi.org/10.4142/jvs.2017.18.2.129

Abstract

To determine heat-shock protein (Hsp)90 expression is connected with cellular apoptotic response to heat stress and its mechanism, chicken (Gallus gallus) primary myocardial cells were treated with the Hsp90 promoter, aspirin, and its inhibitor, geldanamycin (GA), before heat stress. Cellular viability, heat-stressed apoptosis and reactive oxygen species level under different treatments were measured, and the expression of key proteins of the signaling pathway related to Hsp90 and their colocalization with Hsp90 were detected. The results showed that aspirin treatment increased the expression of protein kinase B (Akt), the signal transducer and activator of transcription (STAT)-3 and p-IKKα/β and the colocalization of Akt and STAT-3 with Hsp90 during heat stress, which was accompanied by improved viability and low apoptosis. GA significantly inhibited Akt expression and p-IKKα/β level, but not STAT-3 quantity, while the colocalization of Akt and STAT-3 with Hsp90 was weakened, followed by lower cell viability and higher apoptosis. Aspirin after GA treatment partially improved the stress response and apoptosis rate of tested cells caused by the recovery of Akt expression and colocalization, rather than the level of STAT-3 (including its co-localization with Hsp90) and p-IKKα/β. Therefore, Hsp90 expression has a positive effect on cellular capacity to resist heat-stressed injury and apoptosis. Moreover, inhibition of Hsp90 before stress partially attenuated its positive effects.

Keyword

Hsp90 heat-shock proteins; anti-apoptosis; chicken primary myocardial cells; heat stress disorders

MeSH Terms

Animals
Apoptosis/*physiology
Aspirin/pharmacology
Benzoquinones/pharmacology
Blotting, Western/veterinary
Chick Embryo/cytology
Flow Cytometry/veterinary
HSP90 Heat-Shock Proteins/agonists/antagonists & inhibitors/*metabolism/physiology
Heat-Shock Response/*physiology
In Vitro Techniques
Lactams, Macrocyclic/pharmacology
Myocardium/cytology/*metabolism
Reactive Oxygen Species/metabolism
Benzoquinones
HSP90 Heat-Shock Proteins
Lactams, Macrocyclic
Reactive Oxygen Species
Aspirin

Figure

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  Fig. 1 Effect of heat stress on chicken primary myocardial cells. (A) The effect of heat stress (HS) on cell viability. (B) The effect of HS on cell apoptosis rate. (C) The effect of HS on cellular reactive oxygen species (ROS) level. The difference of the data at different times vs. that at 0 h is indicated by *p < 0.05 and **p < 0.01. The difference of the data at 1 h, 2 h, 3 h vs. that at 5 h and the data at 1 h, 2 h, 3 h vs. that at 7 h are indicated by ††p < 0.01. OD, optical density.

• 

  Fig. 2 The induction of Hsp90 by aspirin (acetylsalicylic acid [ASA]) in chicken primary myocardial cells. (A) The effect of ASA on cell viability. (B) The effect of ASA at different concentrations on Hsp90 level of chicken primary myocardial cells. (C) The effect of ASA at 1 mg/mL on Hsp90 level of chicken primary myocardial cells. (A and B) The difference of data at different concentrations vs. that at 0 mg/mL is indicated by *p < 0.05 and by **p < 0.01. (C) The difference of the data at different times vs. that at 0 h is indicated by *p < 0.05 and by **p < 0.01. OD, optical density.

• 

  Fig. 3 Inhibitory effect of geldanamycin (GA) on Hsp90 in chicken primary myocardial cells. (A) The effect of GA on cell viability. (B) The effect of GA on Hsp70 and Hsp90 in chicken primary myocardial cells. The difference of the data of cells with different treatments vs. that of untreated cells is indicated by *p < 0.05 and by **p < 0.01. OD, optical density.

• 

  Fig. 4 The translocation of heat shock factor (HSF)-1 of chicken primary myocardial cells in response to geldanamycin (GA) and/or aspirin treatment. Chicken, primary myocardial cells, immunofluorescence staining, HSF-1 (red, TRITC), nuclei (blue, DAPI), HSF-1 and nuclei (pink, merged). ASA, acetylsalicylic acid. Scale bar = 20 µm.

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  Fig. 5 Effect of different treatments on Hsp90 expression in chicken primary myocardial cells exposed to heat stress. The difference of the data of cells with different treatments (excluding the data of untreated cells) vs. that of heat-stressed cells are indicated by *p < 0.05. The difference of the data of cells treated by acetylsalicylic acid (ASA)+heat stress (HS) and geldanamycin (GA)+HS vs. that of cells treated by GA+ASA+HS is indicated by ††p < 0.01.

• 

  Fig. 6 Effect of different treatments on protein kinase B (Akt) and the signal transducer and activator of transcription (STAT)-3 expression in chicken primary myocardial cells exposed to heat stress. The difference of the data of cells with different treatments (excluding the data of untreated cells) vs. that of heat-stressed cells are indicated by **p < 0.01. The difference of the data of cells treated by acetylsalicylic acid (ASA)+heat stress (HS) and geldanamycin (GA)+HS vs. that of cells treated by GA+ ASA+HS is indicated by ††p < 0.01.

• 

  Fig. 7 The effect of different treatments on the co-localization of protein kinase B (Akt) and the signal transducer and activator of transcription (STAT)-3 with Hsp90 in chicken primary myocardial cells exposed to heat stress. Immunocytochemical staining. Hsp90 (red, TRITC), Akt/STAT-3 (green, FITC), nuclei (blue, DAPI), Hsp90 and Akt/STAT-3 (yellow, merged), Hsp90 plus Akt/STAT-3 and nuclei (near white, merged). Scale bars = 20 µm.

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  Fig. 8 The effect of different treatments on the p-IKKα/β level in chicken primary myocardial cells exposed to heat stress. The difference of the data of cells with different treatments (excluding the data of untreated cells) vs. that of heat-stressed cells are indicated by *p < 0.05 and by **p < 0.01. The difference between the data of cells treated by acetylsalicylic acid (ASA)+heat stress (HS) and geldanamycin (GA)+HS vs. that of cells treated by GA+ASA+HS is indicated by ††p < 0.01.

• 

  Fig. 9 The effect of different treatments on chicken primary myocardial cells exposed to heat stress. (A) The effect of heat stress (HS) on cell viability. (B) The effect of HS on cell apoptosis rate. (C) The effect of HS on cellular reactive oxygen species (ROS) level. The difference of the data of cells with different treatments (excluding the data of untreated cells) vs. that of heat-stressed cells are indicated by *p < 0.05 and by **p < 0.01. The difference of the data of cells treated by acetylsalicylic acid (ASA)+heat stress (HS) and geldanamycin (GA)+HS vs. that of cells treated by GA+ASA+HS is indicated by †p < 0.05 and by ††p < 0.01.

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  Fig. 10 The effect of different treatments on caspase-3, 8 and 9 activities in chicken primary myocardial cells exposed to heat stress. The difference of the data of cells with different treatments (excluding the data of untreated cells) vs. that of heat-stressed cells are indicated by *p < 0.05 and by **p < 0.01. The difference of the data of cells treated by acetylsalicylic acid (ASA)+heat stress (HS) and geldanamycin (GA)+HS vs. that of cells treated by GA+ASA+HS is indicated by †p < 0.05 and by ††p < 0.01.

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