Background

Pre-eclampsia (PE) is a hypertensive disorder of pregnancy, and a significant clinical problem worldwide. Despite decades of research, this disorder remains without an effective intervention, and the mechanism(s) underlying maternal microvascular dysfunction have not been fully defined.

Within vascular smooth muscle cells (VSMCs), the principal pressure-induced vasodilatory pathway relies upon activation of large-conductance Ca²⁺-activated potassium channels (BK_Ca). Activation is dependent on the release of localised Ca²⁺-release events from sarcoplasmic reticulum (SR) ryanodine receptors, termed ‘Ca²⁺ sparks’, which stimulate BK_Ca channels to cause spontaneous transient outward currents (STOCs) and vasorelaxation. Investigation of BK_Ca channels has been proven effective in advancing treatment of hypertension outside of pregnancy; here, the Ca²⁺ spark – BK_Ca axis is assessed in preeclampsia for the first time.

Methods

Omental biopsies were obtained with informed consent during caesarean section from normotensive women, and women with a clinical diagnosis of preeclampsia, at St Mary’s Hospital, Manchester in accordance with ethics committee approval (National Research Committee North-West – Haydock Board 15/NW/0829).

Pressurised omental resistance arteries were imaged by high-speed spinning-disk laser confocal microscopy to assess Ca²⁺ spark frequency at increasing pressures (20-110 mmHg). Caffeine-evoked Ca²⁺ release was measured in a subset of arteries (10 mmol/L; 80 mmHg), and area under curve (AUC) and transient amplitude calculated. A liquid chromatography–selected reaction monitoring–mass spectrometry (LC-SRM-MS) assay was developed to measure Sarcoplasmic/Endoplasmic Reticulum Ca²⁺ ATPase-2 (SERCA2) and unphosphorylated/phosphorylated proteoforms of regulatory phospholamban. Electrophysiology was used to assess spontaneous transient outward currents (STOCs; -60 to 0 mV) in isolated VSMCs.

Results

In microvascular arteries from women with preeclampsia there was reduced Ca²⁺ spark frequency compared to normal pregnancy (0.53 events/second and 1.90 events/second respectively at 80 mmHg, p=7.3×10^-4; N=13-15), which persisted across the range of pressures measured (p=1.1×10^-10, N=6-15). There was also reduced SR Ca²⁺ release in response to caffeine (AUC 3.42 vs 7.70, p=5.1×10^-4; N=12-13) and decreased ratio of SERCA2 to inhibitory unphosphorylated phospholamban (0.94 vs 2.11, p=0.030; N=5-7), implicating increased SERCA2 inhibition. In VSMCs, STOC frequency was reduced in the preeclampsia cohort (0.11 vs 0.49 events/ second at 10 mV, p=7.1×10^-15; N=5-6).

Conclusions

This data has identified fundamental differences in Ca²⁺ – mediated mechanisms of vascular control in preeclampsia. In microvascular arteries from women with preeclampsia, there is a reduction in pressure-induced Ca²⁺ spark frequency and subsequent BK_Ca STOCs. Additionally, there is a reduction in total SR Ca²⁺ store and the ratio of SERCA2 to its inhibitor, unphosphorylated phospholamban, implicating SR Ca²⁺ loading as the cause of reduced Ca²⁺ sparks. These identified VSMC defects may provide novel therapeutic targets to restore physiological control of the vasculature in preeclampsia.