The interactive association between heat shock factor 1 and heat shock proteins in primary myocardial cells subjected to heat stress

Abstract

Heat shock factor 1 (HSF1) is a heat shock transcription factor that rapidly induces heat shock gene transcription following thermal stress. In this study, we subjected primary neonatal rat myocardial cells to heat stress in vitro to create a model system for investigating the trends in expression and association between various heat shock proteins (HSPs) and HSF1 under adverse environmental conditions. After the cells were subjected to heat stress at 42˚C for different periods of time, HSP and HSF1 mRNA and protein levels were detected by qPCR and western blot analysis in the heat-stressed cells. The HSF1 expression levels significantly increased in the cells following 120 min of exposure to heat stess compared to the levels observed at the beginning of heat stress exposure. HSP90 followed a similar trend in expression to HSF1, whereas HSP70 followed an opposite trend. However, no significant changes were observed in the crystallin, alpha B (CRYAB, also known as HSP beta-5) expression levels during the 480‑min period of exposure to heat stress. The interaction between the HSPs and HSF1 was analyzed by STRING 9.1, and it was found that HSF1 interacted with HSP90 and HSP70, and that it did not play a role in regulating CRYAB expression. Based on our findings, HSP70 may suppress HSF1 in rat myocardial cells under conditions of heat stress. Furthermore, our data demonstrate that HSF1 is not the key factor for all HSPs, and this was particularly the case for CRYAB.

Figure 1

Heat shock proteins (HSPs) and heat shock factor (HSF1) protein expression in heat-stressed rat myocardial cells in vitro. HSPs and HSF1 levels were normalized to those of β-actin during 480 min of exposure to heat stress. HSF1 levels increased gradually and significantly from 240 min of exposure and onwards, and remained elevated after 480 min of heat stress. Compared with the control group, HSP90 expression decreased from 10 to 40 min of exposure to heat, and then increased from 60 to 480 min of heat stress. From 10 to 60 min, the overall expression levels of HSP70 decreased compared with those of the control group, but not at 20 and 60 min of exposure. After 120 min of exposure, the level of HSP70 expression remained low until 480 min. There was no significant change in the crystallin, alpha B (CRYAB) levels during the 480 min of exposure to heat stress. The different letters each indicate a different factor as follows: Aa, HSF1; Bb, HSP90; Cc, SP70; Dd, CRYAB. Upper case letters (A, B and C) represent P<0.01; lower case letter (c) represents P<0.05. All the P-values were compared with the control group.

Figure 2

mRNA expression of heat shock proteins (HSPs) and heat shock factor (HSF1) in primary rat myocardial cells in vitro before and after exposure to heat stress. HSPs and HSF1 mRNA levels were normalized to those of β-actin. HSF1 mRNA levels increased rapidly and in a stepwise manner throughout the time course and reached maximal levels (3-fold induction) after 360 min of exposure to heat stress. The mRNA level of HSP90 increased after 10 min of exposure to heat stress, reaching the maximal level after 240 min of exposure. All other periods of exposure to heat stress resulted in significantly higher levels of HSP90 mRNA than the controls. HSP70 mRNA increased significantly from 10 to 360 min of heat stress compared with the control group. Only the level at 480 min was slightly lower than 360 min of heat stress. The transcription levels of crystallin, alpha B (CRYAB) markedly increased in the heat-stressed myocardial cells. (Aa, HSF1; Bb, HSP90; Cc, HSP70; Dd, CRYAB. ABCD, P<0.01). The different letters each indicate a different factor as follows: A, HSF1; B, HSP90; C, HSP70; D, CRYAB. Upper case letters (A, B, C and D) represent P<0.01. P-values were compared with the control group.

Figure 3

The association between heat shock factor 1 (HSF1) and heat shock proteins (HSPs) in rat myocardial cells analyzed with STRING (version 9.1). (A) HSF1 interacts with HSP90 and HSP70. HSF1 binds with HSP90 and HSP70 (blue line), HSF1 and HSP70 are co-expressed (yellow line), and HSF1 activates HSP70 (green line). (B) Thicker lines indicate a stronger interaction between two proteins. In rats, there is more evidence of an association between HSF1 and HSP70 than between HSF1 and HSP90. (C) Co-expression view showing that HSP70 and HSP60 are co-expressed with HSP90. However, in other species (e.g., Homo sapiens), HSF1 is co-expressed with HSP70.

Figure 4

The interaction between heat shock factor (HSF)2 and heat shock proteins (HSPs) analyzed with STRING (version 9.1). (A) HSF2 interacts with HSP90 and HSP70. HSF2 binds with HSP90 and HSP70 (blue line). crystallin, alpha B (CRYAB) does not interact with HSF1 and HSF2; however, it binds with vimentin and desmin. (B) Thicker lines indicate more evidence of interaction between two proteins. In rats, there is more evidence of an association between HSF2 and HSP70 than between HSF2 and HSP90. (C) Co-expression view showing that CRYAB is co-expressed with desmin in rats.