Calcium-activated potassium channels contribute to human coronary microvascular dysfunction after cardioplegic arrest

Abstract

Background: Cardioplegic arrest (CP) followed by reperfusion after cardiopulmonary bypass induces coronary microvascular dysfunction. We investigated the role of calcium-activated potassium (K(Ca)) channels in this dysfunction in the human coronary microvasculature.

Methods and results: Human atrial tissue was harvested before CP from a nonischemic segment and after CP from an atrial segment exposed to hyperkalemic cold blood CP (mean CP time, 58 minutes) followed by 10-minute reperfusion. In vitro relaxation responses of precontracted arterioles (80 to 180 mum in diameter) in a pressurized no-flow state were examined in the presence of K(Ca) channel activators/blockers and several other vasodilators. We also examined expression and localization of K(Ca) channel gene products in the coronary microvasculature using reverse transcriptase-polymerase chain reaction, immunoblot, and immunofluorescence photomicroscopy. Post-CP reperfusion relaxation responses to the activator of intermediate and small conductance K(Ca) channels (IK(Ca)/SK(Ca)), NS309 (10(-5) M), and to the endothelium-dependent vasodilators, substance P (10(-8) M) and adenosine 5diphosphate (10(-5) M), were significantly reduced compared with pre-CP responses (P<0.05, n=8/group). In contrast, relaxation responses to the activator of large conductance K(Ca) channels (BK(Ca)), NS1619 (10(-5) M), and to the endothelium-independent vasodilator, sodium nitroprusside (10(-4) M), were unchanged pre- and post-CP reperfusion (n=8/group). Endothelial denudation significantly diminished NS309-induced vasodilatation and abolished substance P- or adenosine 5 diphosphate-induced relaxation (P<0.05), but had no effect on relaxation induced by either NS1619 or sodium nitroprusside. The total polypeptide levels of BK(Ca), IK(Ca), and SK(Ca) and the expression of IK(Ca) mRNA were not altered post-CP reperfusion.

Conclusions: Cardioplegic arrest followed by reperfusion after cardiopulmonary bypass causes microvascular dysfunction associated with and likely in part due to impaired function of SK(Ca) and IK(Ca) channels in the coronary microcirculation. These results suggest novel mechanisms of endothelial and smooth muscle microvascular dysfunction after cardiac surgery.

Figure 1

Vasoreactivity of human coronary arterioles to the IK/SK channel activator NS309. A, Dose-dependent relaxation of coronary arterioles (n=8) in response to NS309 before CP and Rep and afterward (post-CP Rep); *P<0.05. B, Dose-dependent NS309-mediated relaxation of pre-CP Rep human coronary arterioles pretreated in the absence or presence of TRAM34 and apamin (n=6); **P<0.001. C, Dose-dependent relaxation of pre-CP Rep human coronary arterioles from pre-CP Rep tissues (n=6) with intact or denuded endothelium in response to NS309; **P<0.001. Data are presented as percent relaxation after preconstriction with U46619.

Figure 2

Vasoreactivity of human coronary arterioles to the BK channel activator NS1619. A, Dose-dependent relaxation of coronary arterioles in response to NS1619 either pre-CP Rep or post-CP Rep (n=8/group). B, Dose-dependent NS1619-mediated relaxation of human coronary arterioles from pre-CP Rep atrial tissues pretreated in the absence or presence of iberiotoxin (n=6; **P<0.001). C, Dose-dependent NS1619-induced relaxation of pre-CP Rep human coronary arterioles with intact or denuded endothelium (n=6/group). Data are presented as percent relaxation after preconstriction with U46619.

Figure 3

In vitro response of preconstricted human atrial arterioles to endogenous vasodilators. Arterioles were compared pre-CP Rep and post-CP Rep (n=8/group). Pre-CP Rep arterioles were compared with intact endothelium and after endothelial denudation (n=6/group) as indicated. A, Response to the endothelium-dependent vasodilator ADP. *P<0.05 versus pre-CP Rep; **P<0.001 versus intact pre-CP Rep. B, Response to substance P. *P<0.05 versus pre-CP Rep; *P<0.001 versus pre-CP Rep. C, Response to the endothelium-independent vasodilator, SNP.

Figure 4

A, top left, Representative immunoblot of human coronary microvessels. Lanes 1 to 4 loaded with 40 μg protein were developed for BK-α, BK-β₁, IK_Ca-α, and SK3_Ca-α polypeptides B, top right, Densitometric evaluation of immunoblot band intensity shows unaltered levels of K_Ca polypeptides after CP Rep (n=5/group). C, bottom, Semiquantitative reverse transcriptase-polymerase chain reaction showing steady-state levels of mRNA for IK_Ca and β-actin control in human atrial tissue pre- and CP and Rep.

Figure 5

Immunolocalization of K_Ca channel polypeptides in human coronary microvessels. Vessels were costained for smooth muscle α-actin and either (A) BK_Ca-α or (B) BK_Ca-β_1. Matched negative controls are displayed below each row of primary antibody staining as indicated.