Arginine vasopressin: Critical regulator of circadian homeostasis.

Circadian rhythms optimally regulate numerous physiological processes in an organism and synchronize them with the external environment. The suprachiasmatic nucleus (SCN), the center of the circadian clock in mammals, is composed of multiple cell types that form a network that provides the basis for the remarkable stability of the circadian clock. Among the neuropeptides expressed in the SCN, arginine vasopressin (AVP) has attracted much attention because of its deep involvement in the function of circadian rhythms, as elucidated in particular by studies using genetically engineered mice. This review briefly summarizes the current knowledge on the peptidergic distribution and topographic neuronal organization in the SCN, the molecular mechanisms of the clock genes, and the relationship between the SCN and peripheral clocks. With respect to the physiological roles of AVP and AVP-expressing neurons, in addition to a sex-dependent action of AVP in the SCN, studies using AVP receptor knockout mice and mice genetically manipulated to alter the clock properties of AVP neurons are summarized here, highlighting its importance in maintaining circadian homeostasis and its potential as a target for therapeutic interventions.

An intact pituitary vasopressin system is critical for building a robust circadian clock in the suprachiasmatic nucleus.

The circadian clock is a biological timekeeping system that oscillates with a circa-24-h period, reset by environmental timing cues, especially light, to the 24-h day-night cycle. In mammals, a "central" clock in the hypothalamic suprachiasmatic nucleus (SCN) synchronizes "peripheral" clocks throughout the body to regulate behavior, metabolism, and physiology. A key feature of the clock's oscillation is resistance to abrupt perturbations, but the mechanisms underlying such robustness are not well understood. Here, we probe clock robustness to unexpected photic perturbation by measuring the speed of reentrainment of the murine locomotor rhythm after an abrupt advance of the light-dark cycle. Using an intersectional genetic approach, we implicate a critical role for arginine vasopressin pathways, both central within the SCN and peripheral from the anterior pituitary.

Comprehensive pathological and genetic investigation of three young adult myotonic dystrophy type 1 patients with sudden unexpected death.

OBJECTIVES: The mechanism and pathological substrate of arrhythmogenic events in dystrophic myopathy type 1 (DM1) have not been fully established, especially for patients without progression of motor and/or cardiac disability. Therefore, we aimed to clarify the pathological appearance and genetic factors, other than CTG repeats in DMPK, associated with sudden cardiac death in patients with DM1. METHODS: A pathological investigation including the cardiac conduction system in the heart and whole-exome sequencing was conducted for three young adults (Patient 1; 25-year-old female, Patient 2; 35-year-old female, Patient 3; 18-year-old male) with DM1 who suffered sudden death. RESULTS: Only Patient 1 showed abnormal electrocardiogram findings before death. The pathological investigation showed severe fibrosis of the atrioventricular conduction system in Patient 1 and severe fatty infiltration in the right ventricle in Patient 2. Several minimal necrotic/inflammatory foci were found in both patients. Patient 3 showed no significant pathological findings. A genetic investigation showed CORIN_p.W813* and MYH2_p. R793* in Patient 1, KCNH2_p. V794D and PLEC_p. A4147T in Patient 2, and SCN5A_p.E428K and SCN3B_ p.V145L in Patient 3 as highly possible pathogenic variants. CONCLUSION AND RELEVANCE: The present study showed varied heart morphology in young adults with DM1 and sudden death. Synergistic effects of various genetic factors other than CTG repeats may increase the risk of sudden cardiac death in DM1 patients, even if signs of cardiac and skeletal muscle involvement are mild. Comprehensive genetic investigations, other than CTG repeat assessment, may be useful to estimate the risk of sudden cardiac death in DM1 patients.

Minimal upstream open reading frame of Per2 mediates phase fitness of the circadian clock to day/night physiological body temperature rhythm.

Body temperature in homeothermic animals does not remain constant but displays a regular circadian fluctuation within a physiological range (e.g., 35°C-38.5°C in mice), constituting a fundamental systemic signal to harmonize circadian clock-regulated physiology. Here, we find the minimal upstream open reading frame (uORF) encoded by the 5' UTR of the mammalian core clock gene Per2 and reveal its role as a regulatory module for temperature-dependent circadian clock entrainment. A temperature shift within the physiological range does not affect transcription but instead increases translation of Per2 through its minimal uORF. Genetic ablation of the Per2 minimal uORF and inhibition of phosphoinositide-3-kinase, lying upstream of temperature-dependent Per2 protein synthesis, perturb the entrainment of cells to simulated body temperature cycles. At the organismal level, Per2 minimal uORF mutant skin shows delayed wound healing, indicating that uORF-mediated Per2 modulation is crucial for optimal tissue homeostasis. Combined with transcriptional regulation, Per2 minimal uORF-mediated translation may enhance the fitness of circadian physiology.

An autopsy case of sudden unexpected death of a young adult with progressive intraventricular conduction delay.

Herein, we describe an autopsy case of the sudden unexpected death of a 23-year-old man. Retrospective analysis of electrocardiograms revealed progressive widening of the QRS interval. Autopsy showed mild mitral valve prolapse and hypertrabeculation of the left ventricle. Microscopic examination revealed very scarce but considerable minimal myocardial necrotic foci in the left ventricle, and a marked reduction in conduction fibers in the left branch. These findings may be associated with intraventricular conduction delay. Genetic investigation revealed four rare possibly pathogenic variants, including the Emery-Dreifuss muscular dystrophy-associated genetic variant SYNE2_p.A6155 V that is evaluated as pathogenic by most in silico predictive tools. The other possibly pathogenic variants detected were PLEC_p.P973L, TTN_p.I22171T, and p.A12216T. Although these variants are reported to have uncertain significance in the guidelines of the American College of Medical Genetics and Genomics, progressive conduction delay may have been associated with vulnerability of myocytes due to Emery-Dreifuss muscular dystrophy-associated genetic variants in the present case. Younger individuals with progressive conduction delay may require medical work-up and genetic investigation, even if they have no other clinical signs and no or mild structural heart disease.

Pathological and Comprehensive Genetic Investigation of Autopsy Cases of Idiopathic Bradyarrhythmia.

BACKGROUND: Idiopathic bradyarrhythmia is considered to be due to pathological degeneration of the cardiac conduction system (CCS) during aging. There appears to have been no comprehensive genetic investigations in patients with idiopathic bradyarrhythmia.Methods and Results: Ten autopsy cases with advanced bradyarrhythmia (6 men and 4 women; age: 70-94 years, 81.5±6.9 years; 5 cases each of sinus node dysfunction [SND] and complete atrioventricular block [CAVB]) were genetically investigated by using whole-exome sequencing. Morphometric analysis of the CCS was performed with sex-, age- and comorbidity-matched control cases. As a result, severe loss of nodal cells and distal atrioventricular conduction system were found in SND and CAVB, respectively. However, the conduction tissue loss was not significant in either the atrioventricular node or the proximal bundle of His in CAVB cases. A total of 13 heterozygous potential variants were found in 3 CAVB and 2 SND cases. Of these 13 variants, 4 were missense in the known progressive cardiac conduction disease-related genes: GATA4 and RYR2. In the remaining 9 variants, 5 were loss-of-function mutation with highly possible pathogenicity. CONCLUSIONS: In addition to degenerative changes of selectively vulnerable areas in the heart during advancing age, the vulnerability of the CCS, which may be associated with "rare variants of small effect," may also be a contributing factor to the degeneration of CCS, leading to "idiopathic" bradyarrhythmia.

Nmu/Nms/Gpr176 Triple-Deficient Mice Show Enhanced Light-Resetting of Circadian Locomotor Activity.

The suprachiasmatic nucleus (SCN) is the master circadian clock in mammals and is properly entrained by environmental light cycle. However, the molecular mechanism(s) determining the magnitude of phase shift by light is still not fully understood. The orphan G-protein-coupled receptor Gpr176 is enriched in the SCN, controls the pace (period) of the circadian rhythm in behavior but is not apparently involved in the light entrainment; Gpr176-/- animals display a shortened circadian period in constant darkness but their phase-resetting responses to light are normal. Here, we performed microarray analysis and identified enhanced mRNA expression of neuromedin U (Nmu) and neuromedin S (Nms) in the SCN of Gpr176-/- mice. By generating C57BL/6J-backcrossed Nmu/Nms/Gpr176 triple knockout mice, we noted that the mutant mice had a greater magnitude of phase shift in response to early subjective night light than wildtype mice, while Nmu/Nms double knockout mice as well as Gpr176 knockout mice are normal in the phase shifts induced by light. At the molecular level, Nmu-/-Nms-/-Gpr176-/- mice had a reduced induction of Per1 and cFos mRNA expression in the SCN by light and mildly upregulated circadian expression of Per2, Prok2, Rgs16, and Rasl11b. These expressional changes may underlie the phenotype of the Nmu/Nms/Gpr176 knockout mice. Our data argue that there is a mechanism requiring Nmu, Nms, and Gpr176 for the proper modulation of light-induced phase shift in mice. Simultaneous modulation of Nmu/Nms/Gpr176 may provide a potential target option for modulating the circadian clock.

A light-induced small G-protein gem limits the circadian clock phase-shift magnitude by inhibiting voltage-dependent calcium channels.

Calcium signaling is pivotal to the circadian clockwork in the suprachiasmatic nucleus (SCN), particularly in rhythm entrainment to environmental light-dark cycles. Here, we show that a small G-protein Gem, an endogenous inhibitor of high-voltage-activated voltage-dependent calcium channels (VDCCs), is rapidly induced by light in SCN neurons via the calcium (Ca2+)-mediated CREB/CRE transcriptional pathway. Gem attenuates light-induced calcium signaling through its interaction with VDCCs. The phase-shift magnitude of locomotor activity rhythms by light, at night, increases in Gem-deficient (Gem-/-) mice. Similarly, in SCN slices from Gem-/- mice, depolarizing stimuli induce larger phase shifts of clock gene transcription rhythms that are normalized by the application of an L-type VDCC blocker, nifedipine. Voltage-clamp recordings from SCN neurons reveal that Ca2+ currents through L-type channels increase in Gem-/- mice. Our findings suggest that transcriptionally activated Gem feeds back to suppress excessive light-evoked L-type VDCC activation, adjusting the light-induced phase-shift magnitude to an appropriate level in mammals.

Paradoxical delayed capture proved the dual-loop tachycardia mechanism of a cavotricuspid isthmus-dependent atrial flutter.

A 37-year-old man underwent catheter ablation for a cavotricuspid isthmus-dependent atrial flutter. Two 20-pole deflectable electrode catheters were placed in a parallel position on the tricuspid annulus and right atrial lateral wall. The dual-loop tachycardia mechanism of the atrial flutter was suggested by paradoxical delayed capture of the lateral wall of the right atrium during entrainment pacing from the lateral tricuspid annulus.

Cardiac vagus nerve denervation by pulmonary vein isolation was effective for swallowing-induced atrial tachycardia.

Swallowing-induced atrial tachycardia (SIAT) is a relatively rare arrhythmia. A 56-year-old woman was admitted to treat atrial tachycardia that occurs by not only eating and drinking but also yawning. Both the right and left upper pulmonary veins were suspected as the earliest activation site of the tachycardia and the abnormal activation of ectopies themselves were suppressed after pulmonary vein isolation (PVI). In a 24-hour Holter electrocardiogram, the HF component of the analysis of heart rate variability was suppressed both at 1 day and at 2 years after ablation. In this case, cardiac vagal nerve denervation by PVI was effective for SIAT.

Intracrine activity involving NAD-dependent circadian steroidogenic activity governs age-associated meibomian gland dysfunction.

Canonically, hormones are produced in the endocrine organs and delivered to target tissues. However, for steroids, the concept of tissue intracrinology, whereby hormones are produced in the tissues where they exert their effect without release into circulation, has been proposed, but its role in physiology/disease remains unclear. The meibomian glands in the eyelids produce oil to prevent tear evaporation, which reduces with aging. Here, we demonstrate that (re)activation of local intracrine activity through nicotinamide adenine dinucleotide (NAD+)-dependent circadian 3ß-hydroxyl-steroid dehydrogenase (3ß-HSD) activity ameliorates age-associated meibomian gland dysfunction and accompanying evaporative dry eye disease. Genetic ablation of 3ß-HSD nullified local steroidogenesis and led to atrophy of the meibomian gland. Conversely, reactivation of 3ß-HSD activity by boosting its coenzyme NAD+ availability improved glandular cell proliferation and alleviated the dry eye disease phenotype. Both women and men express 3ß-HSD in the meibomian gland. Enhancing local steroidogenesis may help combat age-associated meibomian gland dysfunction.

Gpr19 is a circadian clock-controlled orphan GPCR with a role in modulating free-running period and light resetting capacity of the circadian clock.

Gpr19 encodes an evolutionarily conserved orphan G-protein-coupled receptor (GPCR) with currently no established physiological role in vivo. We characterized Gpr19 expression in the suprachiasmatic nucleus (SCN), the locus of the master circadian clock in the brain, and determined its role in the context of the circadian rhythm regulation. We found that Gpr19 is mainly expressed in the dorsal part of the SCN, with its expression fluctuating in a circadian fashion. A conserved cAMP-responsive element in the Gpr19 promoter was able to produce circadian transcription in the SCN. Gpr19-/- mice exhibited a prolonged circadian period and a delayed initiation of daily locomotor activity. Gpr19 deficiency caused the downregulation of several genes that normally peak during the night, including Bmal1 and Gpr176. In response to light exposure at night, Gpr19-/- mice had a reduced capacity for light-induced phase-delays, but not for phase-advances. This defect was accompanied by reduced response of c-Fos expression in the dorsal region of the SCN, while apparently normal in the ventral area of the SCN, in Gpr19-/- mice. Thus, our data demonstrate that Gpr19 is an SCN-enriched orphan GPCR with a distinct role in circadian regulation and may provide a potential target option for modulating the circadian clock.

Thermographic imaging of mouse across circadian time reveals body surface temperature elevation associated with non-locomotor body movements.

Circadian clocks orchestrate multiple different physiological rhythms in a well-synchronized manner. However, how these separate rhythms are interconnected is not exactly understood. Here, we developed a method that allows for the real-time simultaneous measurement of locomotor activity and body temperature of mice using infrared video camera imaging. As expected from the literature, temporal profiles of body temperature and locomotor activity were positively correlated with each other. Basically, body temperatures were high when animals were in locomotion. However, interestingly, increases in body temperature were not always associated with the appearance of locomotor activity. Video imaging revealed that mice exhibit non-locomotor activities such as grooming and postural adjustments, which alone induce considerable elevation of body temperature. Noticeably, non-locomotor movements always preceded the initiation of locomotor activity. Nevertheless, non-locomotor movements were not always accompanied by locomotor movements, suggesting that non-locomotor movements provide a mechanism of thermoregulation independent of locomotor activity. In addition, in the current study, we also report the development of a machine learning-based recording method for the detection of circadian feeding and drinking behaviors of mice. Our data illustrate the potential utility of thermal video imaging in the investigation of different physiological rhythms.

Clinicopathological features of clinically undiagnosed sporadic transthyretin cardiac amyloidosis: a forensic autopsy-based series.

OBJECTIVE: To investigate the clinicopathological features of sporadic amyloid transthyretin (ATTR) amyloidosis. METHODS: We evaluated 1698 serial Japanese forensic autopsy patients. The extent and amount of ATTR deposition in the 16 cardiac regions, including the conduction system, were semiquantitatively evaluated. Ward's hierarchical cluster analysis was applied to classify the cases into subgroups. Also, the relationship between ATTR and amyloid atrial natriuretic factor (AANF) was evaluated. RESULTS: Forty-four cardiac ATTR amyloidosis patients (mean age 85.4 ± 5.7 years; 22 men) without history of hereditary polyneuropathy were identified (2.6% of all patients, 8.8% of those aged ≥80 years). All the 44 patients were not in the bedridden state and died-out-of-hospital scenarios. Of these, 10 (23%) were sudden death. Cluster analysis classified the patients into three groups (mild, atria-predominant and the severe deposition group). Amyloid deposition had already started simultaneously from each atrium and ventricle; however, the atrial septum and basilar ventricular septum were the sites that revealed the most frequent deposition. Also, a possible association between ATTR and AANF deposits was identified. CONCLUSIONS: Sporadic ATTR amyloidosis patients might already be susceptible to a risk for sudden death even from an early-phase. Also, ATTR amyloid deposition in such cases might progress with a certain degree of regularity.

Epicardial adipose tissue affects the efficacy of left atrial posterior wall isolation for persistent atrial fibrillation.

BACKGROUND: Epicardial adipose tissue (EAT) contributes to atrial fibrillation (AF). However, its impact on the efficacy of left atrial posterior wall isolation (LAPWI) is unclear. METHODS: Forty-four nonparoxysmal AF patients underwent LAPWI after pulmonary vein isolation. EAT overlap on LAPWI was assessed by fusing computed tomography images with electro-anatomical mapping. RESULTS: During the 21 ± 7 months of follow-up, AF recurred in 10 patients (23%). The total and left atrial EAT volumes were 113 ± 36 and 33 ± 12 cm3, respectively. No differences were found between the AF-free and AF-recurrent groups regarding EAT volume. The EAT overlaps on LAPWI lines and LAPWI area were 1.2 ± 1.0 and 0.5 ± 0.9 cm2 respectively. Although no difference was found between groups regarding the EAT overlap on LAPWI area, the AF-free group had a significantly larger EAT overlap on LAPWI lines (1.4 ± 1.0 vs 0.6 ± 0.6 cm2, P = .014). Multivariate analysis identified EAT overlap on LAPWI lines as an independent predictor of AF recurrence (hazard ratio: 0.399, 95% confidence interval: 0.178-0.891, P = .025). Kaplan-Meier analysis revealed that, during follow-up, 92% of the large EAT overlap group (≥1.0 cm2) and 58% of the small EAT overlap group (<1.0 cm2) remained AF-free (P = .008). CONCLUSIONS: EAT overlap on LAPWI lines is related to a high AF freedom rate. Direct radiofrequency application to EAT overlap may be necessary to suppress AF.

Ripple map guided catheter ablation targeting abnormal atrial potentials during sinus rhythm for non-paroxysmal atrial fibrillation.

BACKGROUND: Abnormal atrial potential (AAP) during sinus rhythm may be a critical ablation target for atrial fibrillation. However, the assessment of local electrograms throughout the left atrium is difficult. Thus, we sought to investigate the effectiveness of Ripple map guided AAP ablation. METHODS AND RESULTS: AAP areas were determined by Ripple mapping on the CARTO system in 35 patients (Ripple group) by marking the area where small deflections persisted after the first deflection wavefront had passed. Following pulmonary vein isolation, AAP areas were ablated. If AAP areas were located on the left atrial posterior wall, the posterior wall was isolated. The outcome of this approach was compared with that of 66 patients who underwent an empirical linear ablation approach (control group). There were no differences in patient characteristics between the groups. The total radiofrequency application time and procedure time were shorter in the Ripple group than in the control group (radiofrequency application time, 48 ± 14 minutes vs 61 ± 13 minutes, P < .001; procedure time, 205 ± 30 minutes vs 221 ± 27 minutes, P = .013). Gastroparesis occurred in one patient in each group (P = .645), but in both cases this was relieved with conservative therapy. Kaplan-Meier analysis revealed that rate of freedom from atrial arrhythmia was higher in the Ripple group than in the control group (91% vs 74% during the 12 months' follow up; P = .040). CONCLUSION: Ripple map guided AAP ablation effectively suppressed atrial arrhythmia in patients with non-paroxysmal AF.

Identification and functional characterisation of N-linked glycosylation of the orphan G protein-coupled receptor Gpr176.

G-protein-coupled receptors (GPCRs) are important drug targets with diverse therapeutic applications. However, there are still more than a hundred orphan GPCRs, whose protein functions and biochemical features remain unidentified. Gpr176 encodes a class-A orphan GPCR that has a role in circadian clock regulation in mouse hypothalamus and is also implicated in human breast cancer transcriptional response. Here we show that Gpr176 is N-glycosylated. Peptide-N-glycosidase treatment of mouse hypothalamus extracts revealed that endogenous Gpr176 undergoes N-glycosylation. Using a heterologous expression system, we show that N-glycosylation occurs at four conserved asparagine residues in the N-terminal region of Gpr176. Deficient N-glycosylation due to mutation of these residues reduced the protein expression of Gpr176. At the molecular function level, Gpr176 has constitutive, agonist-independent activity that leads to reduced cAMP synthesis. Although deficient N-glycosylation did not compromise this intrinsic activity, the resultant reduction in protein expression was accompanied by attenuation of cAMP-repressive activity in the cells. We also demonstrate that human GPR176 is N-glycosylated. Importantly, missense variations in the conserved N-glycosylation sites of human GPR176 (rs1473415441; rs761894953) affected N-glycosylation and thereby attenuated protein expression and cAMP-repressive activity in the cells. We show that N-glycosylation is a prerequisite for the efficient protein expression of functional Gpr176/GPR176.

P-wave vector magnitude predicts the left atrial low-voltage area in patients with paroxysmal atrial fibrillation.

BACKGROUND: P-wave amplitude (PWA) parameters can be the surrogate measures of the left atrial low-voltage areas (LVAs). METHODS: We measured PWAs using an automated system in 50 patients with paroxysmal atrial fibrillation (AF). We examined the relationships between left atrial LVAs and PWA parameters, including P-wave vector magnitude, calculated as the square root of the sum of lead II PWA squared, lead V6 PWA squared, and a one-half lead V2 PWA squared. RESULTS: Lead I PWA was most strongly correlated with LVAs in the anterior wall and appendage (anterior wall, R = -0.391, P = 0.006; appendage, R = -0.342, P = 0.016), whereas lead II PWA was most strongly correlated with LVAs in the septum, posterior wall, and bottom wall (septum, R = -0.413, P = 0.003; posterior wall, R = -0.297, P = 0.039; bottom wall; R = -0.288, P = 0.045). Although maximum, minimum, mean, and lead I PWAs were not correlated with total LVA, P-wave vector magnitude and lead II PWA were significantly correlated with total LVA (P-wave vector magnitude, R = -0.430, P = 0.002; lead II PWA, R = -0.323, P = 0.023). P-wave vector magnitude achieved the highest accuracy for predicting significant LVA (total LVA > 10%) with an area under the curve of 0.772; sensitivity, specificity, and positive and negative predictive values were 64%, 88%, 85%, and 69%, respectively, for the cutoff value of 0.130 mV. CONCLUSION: P-wave vector magnitude is a useful electrocardiographic predictor of left atrial LVAs.