Large conductance calcium-activated potassium channels contribute to the reduced myogenic tone of peripheral microvasculature after cardiopulmonary bypass

Abstract

Background: We investigated the role of calcium-activated potassium (K(Ca)) channel activation in myogenic tone in human peripheral microvasculature after heart surgery.

Methods: Human skeletal muscle arterioles (90-180microm diameter) were dissected from tissue harvested pre- and post-cardiopulmonary bypass (CPB) during cardiac surgery. Myogenic reactivity in response to stepwise increases in intraluminal pressure was studied between pressure steps. Microvessel tone was determined pre-CPB, post-CPB, and after blockade of K(Ca) channels. Expression and localization of large conductance (BK) K(Ca) channels in the coronary microvasculature was assessed by immunoblot and immunofluorescence photomicroscopy.

Results: Myogenic tone of skeletal muscle arterioles was significantly reduced post-CPB compared with pre-CPB. Decrease in myogenic tone after CPB was reflected by the increase in microvessel internal diameter. Myogenic tone of post-CPB microvessels was significantly increased after treatment with BK(Ca)-blocker iberiotoxin, but unchanged in the combined presence of the blockers of intermediate (IK(Ca)) and small conductance (SK(Ca)) K(Ca) channels, TRAM34/apamin. The increases in myogenic tone after iberiotoxin treatment were demonstrated as a decrease in microvessel internal diameter. No significant differences in BK(Ca) protein levels were noted comparing pre- and post-CPB conditions judged by immunoblot and by immunofluorescence staining of skeletal muscle microvessels. Prominent staining for BK(Ca)-alpha and BK(Ca)-beta(1) subunits localized to the microvascular smooth muscle.

Conclusion: CPB-associated decrease in peripheral myogenic reactivity is likely due to activation of BK(Ca), but not IK(Ca) or SK(Ca). CPB may increase BK(Ca) activity without increasing BK polypeptide level.

FIG. 1

Effects of intraluminal pressure on normalized intraluminal diameter. (A) pre-CPB vessels versus post-CPB; (B) post-CP vessels treated with absence or presence of Iberiotoxin (IBTX), a selective BK_Ca blocker; (C) post-CP vessels treated with absence or presence of the IK_Ca blocker TRAM34 and the SK_Ca blocker apamin (n = 8/group). *P < 0.05 versus pre-CPB or post-CPB, respectively.

FIG. 2

Left: Representative immunoblots of human skeletal muscle microvessels (40 µg loaded protein) developed with antibodies to BK_Ca-α; right: densitometric evaluation of immunoblot band intensity normalized to pre-CPB values shows unaltered levels of BK_Ca-α polypeptides after CPB (n = 6).

FIG. 3

Immunolocalization of BK_Ca-α channel polypeptides inhumanskeletal microvessels. Vessels were co-stained for smooth muscle α-actin and BK_Ca-α. Matched negative controls are displayed below each row of primary antibody staining as indicated. Merged images are at right. (Color version of figure is available online.)

FIG. 4

Immunolocalization of BK_Ca-β₁ channel polypeptides in human skeletal microvessels. Vessels were co-stained for smooth muscle α-actin and BK_Ca-β₁. Matched negative controls are displayed below each row of primary antibody staining as indicated. Merged images are at right. (Color version of figure is available online.)