Comparative analysis of αB-crystallin expression in heat-stressed myocardial cells in vivo and in vitro

Abstract

Relationships between αB-crystallin expression patterns and pathological changes of myocardial cells after heat stress were examined in vitro and in vivo in this study using the H9C2 cell line and Sprague-Dawley rats, respectively. Histopathological lesions, characterized by acute degeneration, karyopyknosis and loss of a defined nucleus, became more severe in rat hearts over the course of heat stress treatment from 20 min to 100 min. The expression of αB-crystallin in rat hearts showed a significant decrease (P<0.05) throughout the heat stress treatment period, except at the 40 min time point. Likewise, decreased αB-crystallin expression was also observed in the H9C2 cell line exposed to a high temperature in vitro, although its expression recovered to normal levels at later time points (80 and 100 min) and the cellular damage was less severe. The results suggest that αB-crystallin is mobilized early after exposure to a high temperature to interact with damaged proteins but that the myocardial cells cannot produce sufficient αB-crystallin for protection against heat stress. Lower αB-crystallin expression levels were accompanied by obvious cell/tissue damage, suggesting that the abundance of this protein is associated with protective effects in myocardial cells in vitro and in vivo. Thus, αB-crystallin is a potential biomarker of heat stress.

Figure 1. Histopathological lesions of rat hearts heat-stressed in vivo.

H&E staining, scale bar = 10 µm. (a) No obvious pathological changes were observed in control rats. In heat-stressed rat hearts, the following were observed at the indicated time points: (b) 20 min, acute granular degeneration (▴) with enlargement of heart cells(♦); (c) 40 min, swollen myocardial cells with reduced nuclei and cloudy cytoplasm (→♦); (d) 60 min, enlarged myocardial cells with acute granular degeneration (▴), accompanied by bleeding (▾); (e) 80 min, myocardial cells with cloudy cytoplasm and karyopyknosis (→); (f) 100 min, loss of defined nuclei (←) in enlarged myocardial cells. (g) In hearts of dead rats at 80 min of heat stress, pathological lesions, characterized by disordered arrangement of cells and loss of striations, along with karyopyknosis (→) and loss of nuclear definition (←), were observed. (h) In hearts of dead rats at 100 min of heat stress, pathological lesions were observed, characterized by karyopyknosis (→) and granular degeneration (▴), accompanied by bleeding (▾).

Figure 2. Cytopathological lesions of H₉C₂ cells heat-stressed in vitro.

H&E staining, scale bar = 10 µm. (a) No obvious pathological changes were observed in control H₉C₂ cells. In heat-stressed H₉C₂ cells, the following were observed at the indicated time points: (b) 20 min, acute degeneration characterized by enlarged cell size (▴); (c) 40 min, light and clear pink granulation (▾) in the cytoplasm and karyopyknosis (→) was observed; (d) 60 min, cloudy cytoplasm (▴). (e) 80 min, enlarged cell size (▴) and intracellular granules (▾); (f) 100 min, acute granular degeneration characterized by numerous pink granules (▾) and enlarged cell size (▴).

Figure 3. Localization of αB-crystallin in rat heart tissue before and after heat stress in vivo.

Immunohistochemical staining, scale bar = 10 µm, αB-crystallin (brown color, DAB). (a) Positive αB-crystallin signals were localized in the cytoplasm of non-stressed rat heart cells. In heat-stressed rat hearts, the following were observed at the indicated time points: (b) 20 min, αB-crystallin signals were positive mainly in the cytoplasm but weaker compared to the control group; (c) 40 min, αB-crystallin signals were still detected in the cytoplasm but stronger than at 20 min of heat stress; (d) 60 min, αB-crystallin was still localized in the cytoplasm; (e) 80 min, weaker positive αB-crystallin signals were still mainly expressed in the cytoplasm; (f) 100 min, αB-crystallin was expressed weakly in the cytoplasm.

Figure 4. Localization of αB-crystallin in H₉C₂ cells before and after heat stress in vitro.

Immunocytochemical staining, scale bar = 10 µm. αB-crystallin (green color, FITC), nuclei (blue color, DAPI) (a) αB-crystallin was localized in the cytoplasm of non-stressed H₉C₂ cells. In heat-stressed H₉C₂ cells, the following were observed at the indicated time points: (b) 20 min, weaker positive αB-crystallin signals localized in the cytoplasm, compared with those of control group; (c) 40 min, αB-crystallin signals localized weakly in the cytoplasm; (d) 60 min, αB-crystallin signals were persistently weaker both in the cytoplasm and over the nucleus; (e) 80 min, αB-crystallin still localized over the nucleus and in the cytoplasm at a lower level; (f) 100 min, most strongly positive αB-crystallin signals localized in the cytoplasm as well as around and over the nucleus.

Figure 5. αB-crystallin expression in rat hearts heat-treated in vivo.

αB-crystallin expression in rat heart tissue decreased (P<0.01) at 20 min of heat stress compared to the control and then significantly increased (P<0.01) at 40 min of heat stress. After 60 min of heat stress, αB-crystallin expression levels decreased again (P<0.01) and remained at lower levels until 100 min of heat stress. However, the level of αB-crystallin in dead rats was much higher than those of the control and all other heat-stressed groups. ** P<0.01; *P<0.05.

Figure 6. αB-crystallin expression in H₉C₂ cells heat treated in vitro.

αB-crystallin decreased significantly (P<0.01) from 40 min to 60 min of heat stress compared to the control group. However, from 80 min to 100 min of heat stress, αB-crystallin levels increased and were not significantly different from that of the control group. ** P<0.01; *P<0.05.