The duper mutation reveals previously unsuspected functions of Cryptochrome 1 in circadian entrainment and heart disease

Abstract

The Cryptochrome 1 (Cry1)-deficient duper mutant hamster has a short free-running period in constant darkness (τ_DD) and shows large phase shifts in response to brief light pulses. We tested whether this measure of the lability of the circadian phase is a general characteristic of Cry1-null animals and whether it indicates resistance to jet lag. Upon advance of the light:dark (LD) cycle, both duper hamsters and Cry1^-/- mice re-entrained locomotor rhythms three times as fast as wild types. However, accelerated re-entrainment was dissociated from the amplified phase-response curve (PRC): unlike duper hamsters, Cry1^-/- mice show no amplification of the phase response to 15' light pulses. Neither the amplified acute shifts nor the increased rate of re-entrainment in duper mutants is due to acceleration of the circadian clock: when mutants drank heavy water to lengthen the period, these aspects of the phenotype persisted. In light of the health consequences of circadian misalignment, we examined effects of duper and phase shifts on a hamster model of heart disease previously shown to be aggravated by repeated phase shifts. The mutation shortened the lifespan of cardiomyopathic hamsters relative to wild types, but this effect was eliminated when mutants experienced 8-h phase shifts every second week, to which they rapidly re-entrained. Our results reveal previously unsuspected roles of Cry1 in phase shifting and longevity in the face of heart disease. The duper mutant offers new opportunities to understand the basis of circadian disruption and jet lag.

Keywords: Cryptochrome 1; cardiomyopathy; circadian rhythms; entrainment; jet lag.

Fig. 1.

Duper hamsters rapidly re-entrain to shifted LD cycles. (A and B) Representative double-plotted actograms of wheel-running activity of wild type (Top) and duper (Bottom) hamsters subjected to abrupt 8-h shifts of the 14L:10D schedule. Yellow shading indicates the time of lights on. (C) Mean (± SEM) latency to establish steady phase angle after an 8-h advance or delay of the 14L:10D cycle. Wild-type hamsters (n = 6) took significantly longer than duper mutants (n = 7) to re-entrain to an 8-h phase advance or an 8-h phase delay (F_3,16 = 34.82, ****P < 0.0001). Wild-type hamsters took longer to re-entrain to a phase delay than a phase advance (***P < 0.002). (D and E) Genotype, but not direction or time of day of the phase shift, determines the latency to re-entrain locomotor activity rhythms. Duper hamsters completed a phase advance more quickly than wild types regardless of whether the shift was instituted by shortening the dark phase (n = 42 dupers, n = 24 wild types) or shortening the light phase (n = 91 dupers, n = 38 wild types). Similarly, dupers completed a phase delay more quickly than wild types regardless of whether the dark phase or the light phase was lengthened (n = 35 wild types, n = 70 dupers). Within genotype, latency to re-entrain did not depend upon whether the dark phase or the light phase was altered. Means were compared with multiple-comparisons ANOVA.

Fig. 2.

Cry1^−/− mice show little jet lag after a 6-h phase advance. (A and B) Representative double-plotted actograms of wheel-running activity of Cry1^−/− (Top) and wild-type (Bottom) mice subjected to a 6-h shift of the 12L:12D cycle. After mice re-entrained to the new LD schedule, they were subjected to a second 6-h phase advance and released into DD 2 days later. The red line fitted to free-running onsets was extrapolated to preshift onset in order to assess phase shift over the first 2 d after the second 6-h phase advance. Upon release into DD 2d after the second 6-h phase advance, mean free-running onset was advanced by 0.77 ± 0.29 h in wild-type and 1.65 ± 0.26 h in Cry1^−/− mice (t_16.27 = 2.238, Welch's t-test, *P < 0.05). (C) Free-running period was calculated while animals were in DD. Cry1^−/− mice had a shorter period than wild types (t₂₅ = 10.98, ****P < 0.0001). (D) Wild-type mice (n = 12) took approximately three times longer than Cry1^−/− mice (n = 15) to re-entrain after the first 6-h phase advance (t₂₅ = 6.812, ****P < 0.0001). (E) Phase response of Cry1^−/− mice (orange circles) and wild-type controls (black squares) to 15’ light pulses in DD. (F) Phase responses were binned to 2-h intervals of circadian time during subjective night and represented as boxplots. There were no significant differences between the two genotypes in shift amplitude at any of the circadian times examined.

Fig. 3.

The effect of the duper mutation to shorten the free-running period does not underlie the phase-shifting phenotype. (A) Mean (± SEM) free-running period of duper hamsters consuming tap water (H₂O) (blue, n = 5) or 25% D₂O adulterated water (black, n = 5). D₂O significantly lengthened the free-running period (t₄ = 3.911, ****P < 0.0001). (B) Phase angle of entrainment estimated as time difference in hours between activity onset and lights off in duper hamsters consuming tap water (blue, n = 6) and D₂O adulterated water (black, n = 6). Heavy water significantly shortened the phase angle of entrainment (F_2,15 = 3.736, *P < 0.05). Upon reinstitution of pure water (post-D₂O, cyan, n = 6), phase angle reverted to pre-D₂O values (F_2,15 = 2.963, P > 0.05). (C and D) Representative actograms plotted modulo τ_DD of a duper hamster when consuming tap water (C) and D₂O adulterated water (D) while maintained in DD. The asterisk indicates 15’ light pulse at CT17.5. Lines fit to activity onsets were used to estimate the free-running period and calculate the phase shift that resulted from the light pulse. (E) Phase-shift amplitude (mean ± SEM, in circadian hours) in response to a 15’ light pulse presented at CT17.5 in duper hamsters consuming tap water (H₂O, blue bar, n = 5) or water adulterated with D₂O (gray bar, n = 5). (F) Latency to re-entrain to 8-h phase advance of the 14L:10D cycle in duper hamsters before (blue bar, n = 6), during (black bar, n = 6), or after (cyan bar, n = 6) consumption of drinking water adulterated with D₂O. Although heavy water slowed the circadian clock of duper hamsters, it did not alter either the amplitude of phase shifts in response to light pulses in the mid subjective night or the rate of re-entrainment after an 8-h shift of the 14L:10D cycle.

Fig. 4.

Phase shifts of the 14L:10D cycle eliminate the effect of the duper mutation to shorten the lifespan of cardiomyopathic (dsg^−/−) hamsters. (A) Kaplan–Meier survival curve of unshifted duper dsg^+/+ and LVG wild-type dsg^+/+ hamsters demonstrates that the duper mutation does not impact longevity in noncardiomyopathic hamsters (P > 0.79, Gehan-Breslow-Wilcoxon test, duper n = 17, wild-type n = 8). Animals that are still living (three duper dsg^+/+ and three LVG wild-type dsg^+/+) are represented by black ticks. (B) Body weight measured every 50 d reveals that duper dsg^+/+ are an average 25 ± 0.01% lighter than LVG wild-type dsg^+/+ (****P < 0.0001, mixed-effects model) (C) Kaplan–Meier survival curve of unshifted duper dsg^−/− and wild-type dsg^−/− hamsters. Cardiomyopathic duper hamsters have a significantly reduced lifespan compared with their wild-type counterparts (****P < 0.0001, Gehan-Breslow-Wilcoxon test, duper n = 33, wild-type n = 35). (D) Kaplan–Meier survival curve of duper dsg^−/− and Bio 14.6 wild-type dsg^−/− hamsters subjected to alternating 8-h advances and delays at 2-wk intervals. Unlike unshifted hamsters of the same genotype, lifespans of duper and wild-type cardiomyopathic hamsters do not differ (P > 0.48, Gehan-Breslow-Wilcoxon test, duper n = 34, wild-type n = 41). (E) Rate of re-entrainment of 8-h shifted hamsters. Duper dsg^−/− hamsters re-entrain faster than wild-type dsg^−/− after both 8-h advances (****P < 0.0001) and delays (****P < 0.0001). Wild-type dsg^−/− hamsters re-entrain faster after 8-h delays than wild-type dsg^+/+ hamsters (****P < 0.0001) but not after 8-h advances (P > 0.07). Duper dsg^−/− hamsters re-entrain faster after 8-h advances than after 8-h delays (****P < 0.0001), although they do not differ from duper dsg^+/+ hamsters (P ≥ 0.23). (F) %EF calculated from M-mode echocardiography of unshifted duper and wild-type dsg^−/− hamsters. Despite the difference in survivorship, %EF declines at a similar rate for both duper and wild-type hamsters, apart from the 210-d time point where the %EF of unshifted duper dsg^−/− hamsters is lower than that of wild-type dsg^−/− (*P = 0.04, Šídák multiple-comparisons test). (G) EF of duper and wild-type dsg^−/− hamsters subjected to alternating 8-h advances and delays. %EF values do not differ between duper and wild type at all timepoints (P > 0.99, Šídák multiple-comparisons test).