Effects of cardiopulmonary bypass on endothelin-1-induced contraction and signaling in human skeletal muscle microcirculation

Abstract

Background: We investigated the effects of cardiopulmonary bypass (CPB) on the contractile response of human peripheral microvasculature to endothelin-1 (ET-1), examined the role of specific ET receptors and protein kinase C-alpha (PKC-α), and analyzed ET-1-related gene/protein expression in this response.

Methods and results: Human skeletal muscle arterioles (90 to 180 μm in diameter) were dissected from tissue harvested before and after CPB from 30 patients undergoing cardiac surgery. In vitro contractile response to ET-1 was assessed by videomicroscopy, with and without an endothelin-A (ET-A) receptor antagonist, an endothelin-B (ET-B) antagonist, or a PKC-α inhibitor. The post-CPB contractile response of peripheral arterioles to ET-1 was significantly decreased compared with pre-CPB response. The response to ET-1 was significantly inhibited in the presence of the ET-A antagonist BQ123 but unchanged in the presence of the ET-B receptor antagonist BQ788. Pretreatment with the PKC-α inhibitor safingol reversed ET-1-induced response from contraction to relaxation. The total protein levels of ET-A and ET-B receptors were not altered after CPB. Microarray analysis showed no significant changes in the gene expression of ET receptors, ET-1-related proteins, and protein kinases after CPB.

Conclusions: CPB decreases myogenic contractile function of human peripheral arterioles in response to ET-1. The contractile response to ET-1 is through activation of ET-A receptors and PKC-α. CPB has no effects on ET-1-related gene/protein expression. These results provide novel mechanisms of ET-1-induced contraction in the setting of vasomotor dysfunction after cardiac surgery.

Figure 1

Skeletal microvascular vasoconstriction in response to endothelin-1 (ET-1) (A) pre- vs. post-cardiopulmonary bypass (CPB), (B) pre-CPB vs. pre-CPB + BQ123, (C) post-CPB vs. post-CPB + BQ123; n = 6–10/group, *P < 0.05 vs. pre-CPB (A, B); or vs. post-CPB (C).

Figure 1

Skeletal microvascular vasoconstriction in response to endothelin-1 (ET-1) (A) pre- vs. post-cardiopulmonary bypass (CPB), (B) pre-CPB vs. pre-CPB + BQ123, (C) post-CPB vs. post-CPB + BQ123; n = 6–10/group, *P < 0.05 vs. pre-CPB (A, B); or vs. post-CPB (C).

Figure 1

Skeletal microvascular vasoconstriction in response to endothelin-1 (ET-1) (A) pre- vs. post-cardiopulmonary bypass (CPB), (B) pre-CPB vs. pre-CPB + BQ123, (C) post-CPB vs. post-CPB + BQ123; n = 6–10/group, *P < 0.05 vs. pre-CPB (A, B); or vs. post-CPB (C).

Figure 2

Skeletal microvascular vasoconstriction in response to endothelin-1 (ET-1) (A) pre-cardiopulmonary bypass (CPB) vs. pre-CPB + BQ788, (B) post-CPB vs. post-CPB + BQ788; n = 6–10/group *P < 0.05 vs. pre-CPB (A) or vs. post-CPB (B).

Figure 2

Skeletal microvascular vasoconstriction in response to endothelin-1 (ET-1) (A) pre-cardiopulmonary bypass (CPB) vs. pre-CPB + BQ788, (B) post-CPB vs. post-CPB + BQ788; n = 6–10/group *P < 0.05 vs. pre-CPB (A) or vs. post-CPB (B).

Figure 3

Skeletal microvascular vasoconstriction in response to Endothelin-1 (ET-1) (A) pre-cardiopulmonary bypass (pre-CPB) vs. pre-CPB + safingol, (B) post-CPB vs. post-CPB + safingol; n= 6–10/group *P < 0.05 vs. pre-CPB (A) or vs. post-CPB (B).

Figure 3

Skeletal microvascular vasoconstriction in response to Endothelin-1 (ET-1) (A) pre-cardiopulmonary bypass (pre-CPB) vs. pre-CPB + safingol, (B) post-CPB vs. post-CPB + safingol; n= 6–10/group *P < 0.05 vs. pre-CPB (A) or vs. post-CPB (B).

Figure 4

Microarry and Immunoblotting data. (A) Microarray analysis showing there were no significant changes of ET-1, ET-A and ET-B receptors gene expression post-CPB. The fold changes were less than 1 for ET-1, ET-A and ET-B receptors; (B) Representative immunoblot of human skeletal microvessels. Lanes 1–2 loaded with 40μg protein were developed for ET-A and ET-B receptor polypeptides (n = 6). Immunoblot band intensity shows unaltered levels of ET-AR and ET-BR polypeptides after CPB.

Figure 4

Microarry and Immunoblotting data. (A) Microarray analysis showing there were no significant changes of ET-1, ET-A and ET-B receptors gene expression post-CPB. The fold changes were less than 1 for ET-1, ET-A and ET-B receptors; (B) Representative immunoblot of human skeletal microvessels. Lanes 1–2 loaded with 40μg protein were developed for ET-A and ET-B receptor polypeptides (n = 6). Immunoblot band intensity shows unaltered levels of ET-AR and ET-BR polypeptides after CPB.

Figure 5

Immunolocalization of ET-A and ET-B receptor polypeptides in human skeletal microvessels (n = 6). Vessels were co-stained for smooth muscle actin and either (A) ET-AR or (B) ET-BR. Matched negative controls are displayed below each row of primary antibody.

Figure 5

Immunolocalization of ET-A and ET-B receptor polypeptides in human skeletal microvessels (n = 6). Vessels were co-stained for smooth muscle actin and either (A) ET-AR or (B) ET-BR. Matched negative controls are displayed below each row of primary antibody.