The first-line cluster headache medication verapamil alters the circadian period and elicits sex-specific sleep changes in mice

Abstract

Verapamil is the first-line preventive medication for cluster headache, an excruciating disorder with strong circadian features. Whereas second-and third-line preventives include known circadian modulators, such as melatonin, corticosteroids, and lithium, the circadian effects of verapamil are poorly understood. Here, we characterize the circadian features of verapamil using both in vitro and in vivo models. In Per2::LucSV reporter fibroblasts, treatment with verapamil (0.03-10 µM) showed a dose-dependent period shortening of the reporter rhythm which reached a nadir at 1 µM, and altered core clock gene expression at 10 µM. Mouse wheel-running activity with verapamil (1 mg/mL added to the drinking water) also resulted in significant period shortening and activity reduction in both male and female free-running wild-type C57BL6/J mice. The temporal patterns of activity reduction, however, differ between the two sexes. Importantly, piezo sleep recording revealed sexual dimorphism in the effects of verapamil on sleep timing and bout duration, with more pronounced adverse effects in female mice. We also found altered circadian clock gene expression in the cerebellum, hypothalamus, and trigeminal ganglion of verapamil-treated mice. Verapamil did not affect reporter rhythms in ex vivo suprachiasmatic nucleus (SCN) slices from Per2:Luc reporter mice, perhaps due to the exceptionally tight coupling in the SCN. Thus, verapamil affects both peripheral (trigeminal ganglion) and central (hypothalamus and cerebellum) nervous system structures involved in cluster headache pathophysiology, possibly with network effects instead of isolated SCN effects. These studies suggest that verapamil is a circadian modulator in laboratory models at both molecular and behavioral levels, and sex is an important biological variable for cluster headache medications. These observations highlight the circadian system as a potential convergent target for cluster headache medications with different primary mechanisms of action.

Clock-Modulating Activities of the Anti-Arrhythmic Drug Moricizine

“…As part of the initial screen [ 37 ], we also found an off-patent anti-arrhythmic drug called moricizine that lengthened the circadian reporter rhythms ( Figure S1A,B ). To further investigate this potential clock-modulating effect, we grew Per2::LucSV reporter cells in 35 mm dishes to confluency, and synchronized by dexamethasone (Dex, 200 nM) or forskolin (Fsk, 1 µM), two commonly used agents for cellular clock synchronization [ 37 , 39 , 40 ]. As shown in Figure 1 A, moricizine was found to lengthen reporter rhythms under both conditions (Dex: DMSO, 23.5 ± 0.1 h vs. moricizine, 25.1 ± 0.1 h; Fsk: DMSO, 23.8 ± 0.05 h vs. moricizine, 24.7 ± 0.05 h).…”

“…To begin to explore the clock-modulatory activities of moricizine in a more physiological setting, we performed tissue explant experiments using Per2::Luc mice as previously described [ 39 , 41 ]. We specifically focused on cardiac tissue explants.…”

“…However, detailed characterization is lacking for most of these agents. We recently characterized the circadian effect of verapamil, a first-line medication for cluster headaches known to display a strong circadian pattern of attacks [ 39 ]. In circadian reporter cells and in wheel-running assays in vivo, we showed that verapamil shortens circadian periods.…”

“…Sample size was based on previous studies [ 39 , 58 ]. Data are presented as mean ± standard deviation (cell culture data) or mean ± standard error of the mean (wheel-running and sleep data).…”

“…Many studies, particularly those on high-throughput chemical screens, have provided evidence that various drugs are able to target circadian oscillators and physiological rhythms in vitro and in vivo, altering circadian period, phase, and amplitude via diverse mechanisms [ 31 , 32 , 33 , 34 , 35 , 36 ]. Previously, we performed several studies to discover and characterize chemical compounds, including drugs, that manipulate circadian oscillators [ 37 , 38 , 39 ]. In an initial chemical screen using the Per2::LucSV reporter fibroblast cells [ 37 ], we identified moricizine as a clock-modulatory drug that alters the reporter circadian rhythms.…”