The changes of vaccinia related kinase 1 in grafted heart after rat heart transplantation

Abstract

Objective: To assess the expression and significance of vaccinia-related kinase 1 (VRK1) after rat heart transplantation.

Materials and methods: Lewis and Wistar rats weighing 250 to 300 g were used as donors and recipients. Allografts were from Wistar transplanted into Lewis, and isografts were transplanted from Lewis into Lewis. Grafts were harvested at 1, 3, 5, and 7 days after transplantation. We performed Western Blot of heart tissues after cardiac transplantation. To analyze VRK1 express between the isografts and allografts for immunohistochemical staining. At 5th day after heart transplantation use related cytokines VRK1 for immunohistochemical. We used double immunofluorescent staining on transverse cryosections of graft tissues by co-labeling with different markers, including those for VRK1, activate caspase-3, α-actinin, VCAM-1, CD4.

Results: Compared with rare expression in syngeneic Lewis rat hearts, VRK1 protein level in allogeneic hearts were detected at various survival times after heterotopic heart transplantation, which observably expressed on day 5 postoperative. In addition, we examined the expression of activate caspase-3 in allogeneic hearts, which has a similar expression with VRK1. Immunohistochemical and immunofluorescent method displayed that VRK1 was widely expressed in cytoplasm of cardiac tissue and activate caspase-3 was also expressed in cardiomyocytes. However, the VRK1 wasn't express in inflammation.

Conclusions: The VRK1 expression has increased after heart transplantation in allograft and isograft, and VRK1 may play a significant role in myocardial apoptosis after heterotopic heart transplantation in rats.

Keywords: Vaccinia-related kinase 1 (VRK1); apoptosis; heterotopic heart transplantation; rat.

Figure 1

The average survival rate of heterotopic heart transplantation. Compared with the isograft, allograft showed no significant difference in average survival rate. (92.5% vs. 88.5%, P>0.05).

Figure 2

Western blot analysis shows that the protein expression of VRK1 in allografts and isografts. The VRK1 expression had increased after heart transplantation. GAPDH was used as an internal control. VRK1, vaccinia-related kinase 1.

Figure 3

The tendency of VRK1 level expression in allografts and isografts. The bar chart below demonstrates the ratio of VRK1 relative to GAPDH expression for each time point. The data are represented as the mean ± SEM (n=5; *, P<0.05). VRK1, vaccinia-related kinase 1.

Figure 4

Immunohistochemical staining of VRK1 in allogeneic and syngeneic cardiac tissue. In the isogeneic transplanted heart, positive VRK1 staining was relatively low, however, in allogeneic transplanted heart the stainings of VRK1-positive cells were increased around the cardiomyocytes, especially at POD5. (Original magnification ×40). VRK1, vaccinia-related kinase 1; POD, postoperative days.

Figure 5

Compared with the syngeneic cardiac graft, the stainings of VRK1-positive cells were increased observably after operation in allografts. Quantitative analysis of VRK1 positive cells after heart transplantation. The VRK1 protein has maximal protein expression at day 5. The data are represented as the mean ± SEM (n=5; *, P<0.05). VRK1, vaccinia-related kinase 1.

Figure 6

Association of VRK1 with α-actinin, and inflammatory cells after heart transplantation. (A,B) The co-localization of VRK1 with α-actinin was observed at POD5; (C) the co-localization of VRK1 with VCAM-1 was not observed at the 5 d in the transplanted heart after operation; (D) the result of co-localization between VRK1 and CD4 could not be observed. (Original magnification ×40). VRK1, vaccinia-related kinase 1; POD, postoperative days.

Figure 7

Quantitative analysis of cardiomyocytes positive cells expressing VRK1 (%) in the isograft and allograft group. *, indicate significant difference at P<0.05 compared with isograft-controlled group. Error bars are represented as the mean ± SEM (n=5; *, P<0.05). VRK1, vaccinia-related kinase 1.

Figure 8

Western blot analysis shows that the protein expression of active caspase-3 in allografts. The active caspase-3 expression had increased after heart transplantation. The changes of active caspase-3 expression were similar to VRK1. VRK1, vaccinia-related kinase 1.

Figure 9

Association of active caspase-3 with VRK1 after heart transplantation. The co-localization of VRK1 with activated caspase-3 was observed at POD5. Representative photomicrographs that illustrate the VRK1 regulate the cardiomyocytes apoptosis after heart transplantation at POD5. (Original magnification ×40). VRK1, vaccinia-related kinase 1; POD, postoperative days.