Skeletal muscle microvasculature response to β-adrenergic stimuli is diminished with cardiac surgery

Abstract

Background: Cardiac surgery and cardiopulmonary bypass are associated with alterations in blood pressure in the perioperative period, which, if uncontrolled, can result in end organ damage or dysfunction. Microvessels, significant contributors to blood pressure, both in the myocardium and peripheral skeletal muscle, have diminished responsiveness to major mediators of vascular tone, including thromboxane and serotonin after cardiopulmonary bypass. Responsiveness of these vessels to β-adrenergic stimulation, a major mediator of vascular tone, has not yet been studied. In this report, we investigated the role of β-adrenergic receptors in vascular tone regulation in human skeletal muscle microvessels before and after β-adrenergic stimulation.

Methods: Skeletal muscle microvessels were isolated from patients undergoing cardiac surgery before and after cardiopulmonary bypass. Vessels were exposed in an ex vivo model to the β-adrenergic agonist isoproterenol, or the direct adenylyl cyclase activator, forskolin, and the selective β-receptor antagonist ICI18.551 hydrochloride plus isoproterenol. Immunofluorescence of β receptors and Western blotting were also performed.

Results: Microvessels showed diminished responsiveness to isoproterenol (10^-6 to 10^-4M) after cardiopulmonary bypass (n = 8/group, P = .01). Pretreatment with the selective β-2 blocker ICI18.551 (10^-6M) prevented isoproterenol-induced microvascular relaxation (P = .001). Forskolin-induced relaxation response was also significantly diminished after cardiopulmonary bypass (n = 4/group, P < .05 versus before cardiopulmonary bypass). No significant changes in the total protein expression of β-1, β-2, and β-3 receptors were detected by western blotting or immunofluorescence.

Conclusion: Microvessels isolated from human skeletal muscle show diminished responsiveness to isoproterenol and its downstream activator forskolin after cardiopulmonary bypass, suggesting there is an alteration in β-adrenergic receptor responsive in adenylate cyclase. The relaxation response to isoproterenol was via activation β-2 receptors without changes in β-adrenergic receptor abundance.

Figure 1.

A. Dose dependent relaxation response to the b-adrenergic receptor agonist isoproterenol (10⁻⁹-10⁻⁴M) pre and post CPB; *p<0.05 vs. pre CPB; B. Dose dependent relaxation response to isoproterenol in the presence or absence of the selective b-2 receptor antagonist ICI18.551 (10⁻⁶M) pre- and post CPB; p<0.05 vs. pre or post CPB alone; C. Dose dependent relaxation response to the direct adenylyl cyclase activator, forskolin pre and post CPB; n =8/group; Mean ± SEM, *p<0.05 vs.pre CPB.

Figure 1.

A. Dose dependent relaxation response to the b-adrenergic receptor agonist isoproterenol (10⁻⁹-10⁻⁴M) pre and post CPB; *p<0.05 vs. pre CPB; B. Dose dependent relaxation response to isoproterenol in the presence or absence of the selective b-2 receptor antagonist ICI18.551 (10⁻⁶M) pre- and post CPB; p<0.05 vs. pre or post CPB alone; C. Dose dependent relaxation response to the direct adenylyl cyclase activator, forskolin pre and post CPB; n =8/group; Mean ± SEM, *p<0.05 vs.pre CPB.

Figure 1.

A. Dose dependent relaxation response to the b-adrenergic receptor agonist isoproterenol (10⁻⁹-10⁻⁴M) pre and post CPB; *p<0.05 vs. pre CPB; B. Dose dependent relaxation response to isoproterenol in the presence or absence of the selective b-2 receptor antagonist ICI18.551 (10⁻⁶M) pre- and post CPB; p<0.05 vs. pre or post CPB alone; C. Dose dependent relaxation response to the direct adenylyl cyclase activator, forskolin pre and post CPB; n =8/group; Mean ± SEM, *p<0.05 vs.pre CPB.

Figure 2.

Immunoblots of human skeletal muscle for β1, β2, and β3 receptors, pre and post CPB. Densitometric analysis shows no significant difference between groups for any receptor between pre and post CPB, n = 6/group, p > 0.05; mean ± standard deviation (SD).

Fig. 3:

A. Representative immunofluorescence of human skeletal muscle for β1, β2, and β3 receptors, alpha smooth muscle actin as a marker of smooth muscle, DAPI for nuclear staining. B. Optical density of fluorescence. No significant difference between pre and post CPB, n = 6/group, p > 0.05; mean ± standard deviation (SD).

Fig. 4:

b-2 adrenergic signaling cascade in smooth muscle. The b-2 receptor is a trans membrane Gs protein coupled receptor. When stimulated by ligands, such as isoproterenol, the alpha subunit’s associated GDP is exchanged for GTP, allowing the subunit to be relased from the receptor complex and associate with adenylyl cyclase. Adenylyl cyclase converts ATP into cyclic AMP, a positive regulator of protein kinase A. Protein kinase A phosphorylates myosin light chain kinase, inactivating it and preventing its phosphorylation of myosin light chain (MLC). In the absence of phosphorylation, MLC is unable to form cross links with actin and the muscle relaxes. In the vasculature, this results in an increased vessel diameter.