Martin R. Ralph

CIRCADIAN RHYTHM DISORGANIZATION PRODUCES PROFOUND CARDIOVASCULAR AND RENAL DISEASE IN HAMSTERS

Sleep deprivation, shift work, and jet lag all disrupt normal biological rhythms and have major impacts on health; however, circadian disorganization has never been shown as a causal risk factor in organ disease. We now demonstrate devastating effects of rhythm disorganization on cardiovascular and renal integrity and that interventions based on circadian principles prevent disease pathology caused by a short-period mutation (tau) of the circadian system in hamsters. The point mutation in the circadian regulatory gene, casein kinase-1epsilon, produces early onset circadian entrainment with fragmented patterns of behavior in +/tau heterozygotes. Animals die at a younger age with cardiomyopathy, extensive fibrosis, and severely impaired contractility; they also have severe renal disease with proteinuria, tubular dilation, and cellular apoptosis. On light cycles appropriate for their genotype (22 h), cyclic behavioral patterns are normalized, cardiorenal phenotype is reversed, and hearts and kidneys show normal structure and function. Moreover, hypertrophy does not develop in animals whose suprachiasmatic nucleus was ablated as young adults. Circadian organization therefore is critical for normal health and longevity, whereas chronic global asynchrony is implicated in the etiology of cardiac and renal disease.

The Significance of Circadian Organization for Longevity in the Golden Hamster

While functional roles for biological clocks have been demonstrated in organisms throughout phylogeny, the adaptive advantages of circadian organization per se are largely matters of conjecture. It is generally accepted, though without direct experimental evidence, that organisms derive primary benefits from the temporal organization of their physiology and behavior, as well as from the anticipation of daily changes in their environment and their own fluctuating physiological requirements. However, the consequences of circadian dysfunction that might demonstrate a primary adaptive advantage and explain the natural origins and apparent ubiquity of circadian systems have not been documented. The authors report that longevity in hamsters is decreased with a noninvasive disruption of rhythmicity and is increased in older animals given suprachiasmatic implants that restore higher amplitude rhythms. The results substantiate the importance of the temporal organization of physiology and behavior provided by the circadian clock to the health and longevity of an organism.

Do NMDA receptors mediate the effects of light on circadian behavior

We report here the results of experiments designed to evaluate whether a specific NMDA receptor antagonist, (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,b]cyclohepten-5,10-imine maleate (MK-801), blocks the phase shifting effects of light on the circadian rhythm of wheel-running activity in golden hamsters. Intraperitoneal administration of (+)-MK-801 produced a dose-dependent blockade of both light-induced phase advances and delays. The effect was stereoselective and treatment with related compounds, phenylcyclidine and ketamine, also blocked light-induced phase shifts. MK-801, by itself, did not cause any consistent effect on the phase of the rhythm. These data, coupled with previous findings, indicate that excitatory amino acid receptors play an important role in the transmission of light information from the retina to the circadian system.

Emergence of Noise-Induced Oscillations in the Central Circadian Pacemaker

Computational modeling and experimentation explain how intercellular coupling and intracellular noise can generate oscillations in a mammalian neuronal network even in the absence of cell-autonomous oscillators.

Disturbed Diurnal Rhythm Alters Gene Expression and Exacerbates Cardiovascular Disease With Rescue by Resynchronization

Day/night rhythms are recognized as important to normal cardiovascular physiology and timing of adverse cardiovascular events; however, their significance in disease has not been determined. We demonstrate that day/night rhythms play a critical role in compensatory remodeling of cardiovascular tissue, and disruption exacerbates disease pathophysiology. We use a murine model of pressure overload cardiac hypertrophy (transverse aortic constriction) in a rhythm-disruptive 20-hour versus 24-hour environment. Echocardiography reveals increased left ventricular end-systolic and -diastolic dimensions and reduced contractility in rhythm-disturbed transverse aortic constriction animals. Furthermore, cardiomyocytes and vascular smooth muscle cells exhibit reduced hypertrophy, despite increased pressure load. Microarray and real-time PCR demonstrate altered gene cycling in transverse aortic constriction myocardium and hypothalamic suprachiasmatic nucleus. With rhythm disturbance, there is a consequent altered cellular clock mechanism (per2 and bmal), whereas key genes in hypertrophic pathways (ANF, BNP, ACE, and collagen) are downregulated paradoxical to the increased pressure. Phenotypic rescue, including reversal/attenuation of abnormal pathology and genes, only occurs when the external rhythm is allowed to correspond with the animals’ innate 24-hour internal rhythm. Our study establishes the importance of diurnal rhythm as a vital determinant in heart disease. Disrupted rhythms contribute to progression of organ dysfunction; restoration of normal diurnal schedules appears to be important for effective treatment of disease.

Behavioral Inhibition of Circadian Responses to Light

Circadian locomotor rhythms in rodents may be synchronized by either photic or nonphotic events that produce phase shifts of the rhythm. Little is known, however, about how these two types of stimuli interact to produce entrainment. The well-characterized circadian photic response of the golden hamster was examined in situations where a short light pulse and locomotor activity, a nonphotic event, occurred simultaneously. Light-induced phase advances were attenuated when animals were active during light exposure. The results show that circadian responses to light depend upon the environmental situation in which the light is given, and call into question the implicit assumption in circadian rhythm research that phase shifting and entrainment to light-dark cycles depend simply on photic activation of well-known retinofugal pathways. Moreover, since light therapy is becoming an important component in the treatment of circadian-based disorders in humans, the results emphasize the need for evaluation of the behavioral aspects of light therapy protocols.

Dexras1 Potentiates Photic and Suppresses Nonphotic Responses of the Circadian Clock

Circadian rhythms of physiology and behavior are generated by biological clocks that are synchronized to the cyclic environment by photic or nonphotic cues. The interactions and integration of various entrainment pathways to the clock are poorly understood. Here, we show that the Ras-like G protein Dexras1 is a critical modulator of the responsiveness of the master clock to photic and nonphotic inputs. Genetic deletion of Dexras1 reduces photic entrainment by eliminating a pertussis-sensitive circadian response to NMDA. Mechanistically, Dexras1 couples NMDA and light input to Gi/o and ERK activation. In addition, the mutation greatly potentiates nonphotic responses to neuropeptide Y and unmasks a nonphotic response to arousal. Thus, Dexras1 modulates the responses of the master clock to photic and nonphotic stimuli in opposite directions. These results identify a signaling molecule that serves as a differential modulator of the gated photic and nonphotic input pathways to the circadian timekeeping system.

Circadian rhythms, aging and memory

In human beings and animal models, cognitive performance is often impaired in natural and experimental situations where circadian rhythms are disrupted. This includes a general decline in cognitive ability and fragmentation of behavioural rhythms in the aging population of numerous species. There is some evidence that rhythm disruption may lead directly to cognitive impairment; however, this causal link has not been made for effects due to aging. We have tested this link by examining rhythms and performance on contextual conditioning with the conditioned place preference task, in elderly, age-matched hamsters. Young healthy hamsters developed a preference for a context that is paired with the opportunity to engage in wheel-running (experiment 1). Aged animals with consolidated locomotor rhythms developed similar degrees of preference, whereas the age-matched hamsters with fragmented rhythms did not (experiment 2). The degree of preference was also correlated with activity amplitude. These results support the notion that age-related rhythm fragmentation contributes to the age-related memory decline.

Mania-like behavior induced by genetic dysfunction of the neuron-specific Na+,K+-ATPase α3 sodium pump.

Bipolar disorder is a debilitating psychopathology with unknown etiology. Accumulating evidence suggests the possible involvement of Na+,K+-ATPase dysfunction in the pathophysiology of bipolar disorder. Here we show that Myshkin mice carrying an inactivating mutation in the neuron-specific Na+,K+-ATPase α3 subunit display a behavioral profile remarkably similar to bipolar patients in the manic state. Myshkin mice show increased Ca2+ signaling in cultured cortical neurons and phospho-activation of extracellular signal regulated kinase (ERK) and Akt in the hippocampus. The mood-stabilizing drugs lithium and valproic acid, specific ERK inhibitor SL327, rostafuroxin, and transgenic expression of a functional Na+,K+-ATPase α3 protein rescue the mania-like phenotype of Myshkin mice. These findings establish Myshkin mice as a unique model of mania, reveal an important role for Na+,K+-ATPase α3 in the control of mania-like behavior, and identify Na+,K+-ATPase α3, its physiological regulators and downstream signal transduction pathways as putative targets for the design of new antimanic therapies.

Day/night rhythms in gene expression of the normal murine heart

Molecular circadian oscillators have recently been identified in heart and many other peripheral organs; however, little is known about the physiologic significance of circadian gene cycling in the periphery. While general temporal profiles of gene expression in the heart have been described under constant lighting conditions, patterns under normal day/night conditions may be distinctly different. To understand how gene expression contributes to cardiac function, especially in human beings, it is crucial to examine these patterns in 24-h light and dark environments. High-density oligonucleotide microarrays were used to assess myocardial expression of 12,488 murine genes at 3-h intervals under the normal conditions of light and dark cycling. Variation in genetic activity was considerable, as 1,634 genes (~13% of genes analyzed) exhibited statistically significant changes across the 24-h cycle. Some genes exhibited rhythmic expression, others showed abrupt change at light-to-dark and dark-to-light transitions. Importantly, genes that exhibited significant cycling rhythms mapped to key biological pathways, including for example cardiac cellular growth and remodeling, as well as transcription, translation, mitochondrial respiration, and signaling pathways. Gene expression in the heart is remarkably different in the day versus the night. Some gene cycling may be driven by the central circadian pacemaker, while other changes appear to be responses to light and dark. This has important implications regarding our understanding of how the molecular physiology of the heart is controlled, including temporal patterns of organ growth, renewal, and disease, comparative gene expression, and the most appropriate times for administration of therapy.