Feeding Behavior Modifies the Circadian Variation in RR and QT intervals by Distinct Mechanisms in Mice.

Rhythmic feeding behavior is critical for regulating phase and amplitude in the ¼24-hour variation of heart rate (RR intervals), ventricular repolarization (QT intervals), and core body temperature in mice. We hypothesized changes in cardiac electrophysiology associated with feeding behavior were secondary to changes in core body temperature. Telemetry was used to record electrocardiograms and core body temperature in mice during ad libitum-fed conditions and after inverting normal feeding behavior by restricting food access to the light cycle. Light cycle-restricted feeding modified the phase and amplitude of 24-hour rhythms in RR and QT intervals, and core body temperature to realign with the new feeding time. Changes in core body temperature alone could not account for changes in phase and amplitude in the ¼24-hour variation of the RR intervals. Heart rate variability analysis and inhibiting ß-adrenergic and muscarinic receptors suggested that changes in the phase and amplitude of 24-hour rhythms in RR intervals were secondary to changes in autonomic signaling. In contrast, changes in QT intervals closely mirrored changes in core body temperature. Studies at thermoneutrality confirmed that the daily variation in QT interval, but not RR interval, primarily reflected daily changes in core body temperature (even in ad libitum-fed conditions). Correcting the QT interval for differences in core body temperature helped unmask QT interval prolongation after starting light cycle-restricted feeding and in a mouse model of long QT syndrome. We conclude feeding behavior alters autonomic signaling and core body temperature to regulate phase and amplitude in RR and QT intervals, respectively.

Circadian Regulation of Cardiac Arrhythmias and Electrophysiology.

Circadian rhythms in physiology and behavior are ≈24-hour biological cycles regulated by internal biological clocks (ie, circadian clocks) that optimize organismal homeostasis in response to predictable environmental changes. These clocks are present in virtually all cells in the body, including cardiomyocytes. Many decades ago, clinicians and researchers became interested in studying daily patterns of triggers for sudden cardiac death, the incidence of sudden cardiac death, and cardiac arrhythmias. This review highlights historical and contemporary studies examining the role of day/night rhythms in the timing of cardiovascular events, delves into changes in the timing of these events over the last few decades, and discusses cardiovascular disease-specific differences in the timing of cardiovascular events. The current understanding of the environmental, behavioral, and circadian mechanisms that regulate cardiac electrophysiology is examined with a focus on the circadian regulation of cardiac ion channels and ion channel regulatory genes. Understanding the contribution of environmental, behavioral, and circadian rhythms on arrhythmia susceptibility and the incidence of sudden cardiac death will be essential in developing future chronotherapies.

Characterization of gene expression in the kidney of renal tubular cell-specific NFAT5 knockout mice.

Nuclear factor of activated T cells 5 (NFAT5; also called TonEBP/OREBP) is a transcription factor that is activated by hypertonicity and induces osmoprotective genes to protect cells against hypertonic conditions. In the kidney, renal tubular NFAT5 is known to be involved in the urine concentration mechanism. Previous studies have suggested that NFAT5 modulates the immune system and exerts various effects on organ damage, depending on organ and disease states. Pathophysiological roles of NFAT5 in renal tubular cells, however, still remain obscure. We conducted comprehensive analysis by performing transcription start site (TSS) sequencing on the kidney of inducible and renal tubular cell-specific NFAT5 knockout (KO) mice. Mice were subjected to unilateral ureteral obstruction to examine the relevance of renal tubular NFAT5 in renal fibrosis. TSS sequencing analysis identified 722 downregulated TSSs and 1,360 upregulated TSSs, which were differentially regulated ≤-1.0 and ≥1.0 in log2 fold, respectively. Those TSSs were annotated to 532 downregulated genes and 944 upregulated genes, respectively. Motif analysis showed that sequences that possibly bind to NFAT5 were enriched in TSSs of downregulated genes. Gene Ontology analysis with the upregulated genes suggested disorder of innate and adaptive immune systems in the kidney. Unilateral ureteral obstruction significantly exacerbated renal fibrosis in the renal medulla in KO mice compared with wild-type mice, accompanied by enhanced activation of immune responses. In conclusion, NFAT5 in renal tubules could have pathophysiological roles in renal fibrosis through modulating innate and adaptive immune systems in the kidney.NEW & NOTEWORTHY TSS-Seq analysis of the kidney from renal tubular cell-specific NFAT5 KO mice uncovered novel genes that are possibly regulated by NFAT5 in the kidney under physiological conditions. The study further implied disorders of innate and adaptive immune systems in NFAT5 KO mice, thereby exacerbating renal fibrosis at pathological states. Our results may implicate the involvement of renal tubular NFAT5 in the progression of renal fibrosis. Further studies would be worthwhile for the development of novel therapy to treat chronic kidney disease.

Feeding Behavior Modifies Daily Rhythms in Autonomic Signaling and Core Body Temperature to Shape 24-hour Rhythms in Cardiac Electrophysiology.

Circadian rhythms in physiology and behavior are intrinsic ~24-hour cycles regulated by biological clocks (i.e., circadian clocks) that optimize organismal homeostasis in response to predictable environmental changes. Studies suggest that circadian clock signaling in the suprachiasmatic nucleus of the hypothalamus and cardiomyocytes shape day/night rhythms in cardiac electrophysiology (i.e., RR and QT intervals). However, studies also show that the day/night rhythm of the RR and QT intervals depends on the timing of feeding in mice. This study determined the mechanisms for how feeding impacts day/night rhythms in the RR and QT intervals in mice. Telemetry was used to record electrocardiograms, core body temperature, and activity in mice during ad libitum-fed conditions and after inverting normal feeding behavior by restricting the timing of feeding to the light cycle. Light-cycle restricted feeding caused a simultaneous realignment of RR, QT, and PR intervals and body temperature to the new feeding time. Correcting the QT interval for body temperature eliminated the 24-hour rhythm in the QT interval. Estimating the impact of temperature on RR intervals did not account for the daily change in the RR interval during light-cycle restricted feeding. Cross-correlation analysis suggested daily rhythm in RR intervals correlated with heart rate variability measures but not activity. Injecting mice undergoing light cycle-restricted feeding with propranolol and atropine caused a complete loss in the 24-hour rhythm in the RR interval. We conclude that feeding behavior impacts body temperature and autonomic regulation of the heart to generate 24-hour rhythms in RR and QT intervals.

An infant with A20 haploinsufficiency presenting with periodic fever syndrome: A case report.

A20 haploinsufficiency (HA20) is an early-onset autoinflammatory disease caused by loss-of-function variants of the TNFAIP3 gene, which encodes the protein A20. HA20 is typically characterized by Behçet's disease-like clinical symptoms, and patients usually present with a family history. Herein, we report a case of HA20 in a pediatric patient, presenting with periodic fever, abdominal pain, and vomiting, with no family history. This patient also harbored a novel heterozygous frameshift variant c.677del (p.Pro226LeufsTer2) of TNFAIP3. We initiated treatment with an anti-tumor necrosis factor-α agent that did not induce symptom resolution; we thus administered combination therapy, including prednisolone. Remission was then successfully achieved. We suggest that HA20 should be considered when an autoinflammatory disease is suspected and periodic fever syndrome is present, even in the absence of a family history of HA20 or Behçet's disease-like symptoms.

FGF9 variant in 46,XY DSD patient suggests a role for dimerization in sex determination.

46,XY gonadal dysgenesis (GD) is a Disorder/Difference of Sex Development (DSD) that can present with phenotypes ranging from ambiguous genitalia to complete male-to-female sex reversal. Around 50% of 46,XY DSD cases receive a molecular diagnosis. In mice, Fibroblast growth factor 9 (FGF9) is an important component of the male sex-determining pathway. Two FGF9 variants reported to date disrupt testis development in mice, but not in humans. Here, we describe a female patient with 46,XY GD harbouring the rare FGF9 variant (missense mutation), NM_002010.2:c.583G > A;p.(Asp195Asn) (D195N). By biochemical and cell-based approaches, the D195N variant disrupts FGF9 protein homodimerisation and FGF9-heparin-binding, and reduces both Sertoli cell proliferation and Wnt4 repression. XY Fgf9D195N/D195N foetal mice show a transient disruption of testicular cord development, while XY Fgf9D195N/- foetal mice show partial male-to-female gonadal sex reversal. In the general population, the D195N variant occurs at an allele frequency of 2.4 × 10-5 , suggesting an oligogenic basis for the patient's DSD. Exome analysis of the patient reveals several known and novel variants in genes expressed in human foetal Sertoli cells at the time of sex determination. Taken together, our results indicate that disruption of FGF9 homodimerization impairs testis determination in mice and, potentially, also in humans in combination with other variants.

The Differential Prognostic Impact of Long-Duration Atrial High-Rate Episodes Detected by Cardiac Implantable Electronic Devices between Patients with and without a History of Atrial Fibrillation.

Long-duration atrial high-rate episodes (AHREs) monitored using cardiac implantable electronic devices (CIEDs) can predict long-term major adverse cardiovascular events (MACEs). This study aimed to compare the impact of long-duration AHRE on MACE development between patients with and without a history of atrial fibrillation (AF). This single-center observational study included 132 CIED-implanted patients with AHREs detected via remote monitoring. The population was dichotomized into groups: with (n = 69) and without (n = 63) AF. In each group, cumulative incidences of MACEs comprising all-cause deaths, heart failure hospitalizations, strokes, and acute coronary syndromes were compared between patients with AHRE durations of ≥24 h and <24 h. Multivariate analysis was performed to identify predictors of MACEs among patients without AF. MACE incidence was significantly higher in patients with AHRE ≥24 h than in those with <24 h in the group without AF (92% vs. 30%, p = 0.005). MACE incidence did not significantly differ between AHRE ≥24 h and <24 h in the group with AF (54% vs. 26%, p = 0.44). After a multivariate adjustment, AHRE duration of ≥24 h emerged as the only independent predictor of MACEs among patients without AF (p = 0.03). In conclusion, a long-duration AHRE was prognostic in patients without a history of AF but not in patients with a history of AHREs.

The role of the cardiomyocyte circadian clocks in ion channel regulation and cardiac electrophysiology.

Daily variations in cardiac electrophysiology and the incidence for different types of arrhythmias reflect ≈24 h changes in the environment, behaviour and internal circadian rhythms. This article focuses on studies that use animal models to separate the impact that circadian rhythms, as well as changes in the environment and behaviour, have on 24 h rhythms in heart rate and ventricular repolarization. Circadian rhythms are initiated at the cellular level by circadian clocks, transcription-translation feedback loops that cycle with a periodicity of 24 h. Several studies now show that the circadian clock in cardiomyocytes regulates the expression of cardiac ion channels by multiple mechanisms; underlies time-of-day changes in sinoatrial node excitability/intrinsic heart rate; and limits the duration of the ventricular action potential waveform. However, the 24 h rhythms in heart rate and ventricular repolarization are primarily driven by autonomic signalling. A functional role for the cardiomyocyte circadian clock appears to buffer the heart against perturbations. For example, the cardiomyocyte circadian clock limits QT-interval prolongation (especially at slower heart rates), and it may facilitate the realignment of the 24 h rhythm in heart rate to abrupt changes in the light cycle. Additional studies show that modifying rhythmic behaviours (including feeding behaviour) can dramatically impact the 24 h rhythms in heart rate and ventricular repolarization. If these mechanisms are conserved, these studies suggest that targeting endogenous circadian mechanisms in the heart, as well as modifying the timing of certain rhythmic behaviours, could emerge as therapeutic strategies to support heart function against perturbations and regulate 24 h rhythms in cardiac electrophysiology.

Impact of Atrial Tachyarrhythmia Recurrence on the Development of Long-Term Adverse Clinical Events Following Catheter Ablation in Patients With Atrial Fibrillation With Systolic Impairment: A Single-Center Observational Study.

Background Catheter ablation can improve long-term prognosis of patients with atrial fibrillation with systolic impairment. However, atrial tachyarrhythmia (ATA) recurrence increases during long-term follow-up. We aimed to investigate the impact of ATA recurrence on the development of long-term adverse clinical events following catheter ablation for atrial fibrillation and to identify predictors for the development of adverse clinical events. Methods and Results This single-center observational study included 75 patients with systolic impairment (left ventricular ejection fraction <50%) who underwent the first catheter ablation procedure for atrial fibrillation at our institution (median follow-up period: 3.5 [range: 2.4-4.7] years). We compared the cumulative incidence of adverse clinical events (all-cause death, heart failure hospitalization, stroke, or acute myocardial infarction) between the groups with and without ATA recurrence following the first and last procedures. Multivariable analyses were performed to identify predictors for developing adverse clinical events. Twenty-one patients (28%) developed adverse clinical events at a median of 2.2 (range: 0.64-2.8) years following the first procedure. The proportion of freedom from adverse clinical events following the first procedure was significantly lower in the ATA recurrence group than in the nonrecurrence group (41% [n=40] versus 95% [n=35], P<0.0005); the proportion following the last procedure also showed a similar tendency (35% [n=26] versus 57% [n=49], P<0.0001). ATA recurrence emerged as an independent predictor for adverse clinical events following both procedures after multivariable adjustment. Conclusions ATA recurrence following catheter ablation procedure could predict adverse clinical events in patients with atrial fibrillation with systolic impairment.

Timing of food intake in mice unmasks a role for the cardiomyocyte circadian clock mechanism in limiting QT-interval prolongation.

Cardiac electrophysiological studies demonstrate that restricting the feeding of mice to the light cycle (time restricted feeding or TRF) causes a pronounced change in heart rate and ventricular repolarization as measured by the RR- and QT-interval, respectively. TRF slows heart rate and shifts the peak (acrophase) of the day/night rhythms in the RR- and QT-intervals from the light to the dark cycle. This study tested the hypothesis that these changes in cardiac electrophysiology are driven by the cardiomyocyte circadian clock mechanism. We determined the impact that TRF had on RR- and QT-intervals in control mice or mice that had the cardiomyocyte circadian clock mechanism disrupted by inducing the deletion of Bmal1 in adult cardiomyocytes (iCSΔBmal1-/- mice). In control and iCSΔBmal1-/- mice, TRF increased the RR-intervals measured during the dark cycle and shifted the acrophase of the day/night rhythm in the RR-interval from the light to the dark cycle. Compared to control mice, TRF caused a larger prolongation of the QT-interval measured from iCSΔBmal1-/- mice during the dark cycle. The larger QT-interval prolongation in the iCSΔBmal1-/- mice caused an increased mean and amplitude in the day/night rhythm of the QT-interval. There was not a difference in the TRF-induced shift in the day/night rhythm of the QT-interval measured from control or iCSΔBmal1-/- mice. We conclude that the cardiomyocyte circadian clock does not drive the changes in heart rate or ventricular repolarization with TRF. However, TRF unmasks an important role for the cardiomyocyte circadian clock to prevent excessive QT-interval prolongation, especially at slow heart rates.

An impact of superior vena cava isolation in non-paroxysmal atrial fibrillation patients with low voltage areas.

BACKGROUND: This study aimed to investigate the correlation between left atrial low-voltage areas (LVAs) and an arrhythmogenic superior vena cava (SVC) and the impact on the efficacy of an empiric SVC isolation (SVCI) along with a pulmonary vein isolation (PVI) of non-paroxysmal atrial fibrillation (non-PAF) with or without LVAs. METHODS: We retrospectively enrolled 153 consecutive patients with non-PAF who underwent a PVI alone (n = 51) or empiric PVI plus an SVCI (n = 102). Left atrial voltage maps were constructed during sinus rhythm to identify the LVAs (<0.5 mV). An arrhythmogenic SVC was defined as firing from the SVC and an SVC associated with the maintenance of AF-like rapid SVC activity. RESULTS: An arrhythmogenic SVC and LVAs were identified in 28% and 65% of patients with a PVI alone and 36% and 73% of patients with a PVI plus SVCI, respectively (P = .275 and P = .353). In the multivariate analysis a female gender, higher pulmonary artery systolic pressure (PAPs), and arrhythmogenic SVC were associated with the presence of LVAs. In the PVI plus SVCI strategy, there was no significant difference in the atrial tachyarrhythmia/AF-free survival between the patients with and without LVAs after initial and multiple sessions (50% vs. 61%; P = .386, 73% vs. 79%; P = .530), however, differences were observed in the PVI alone group (27% vs. 61%; P = .018, 49% vs. 78%; P = .046). CONCLUSIONS: The presence of LVAs was associated with an arrhythmogenic SVC. An SVCI may have the potential to compensate for an impaired outcome after a PVI in non-PAF patients with LVAs.

Pentafluoroethylation of Carbonyl Compounds Using HFC-125 in a Flow Microreactor System.

The protocol of micro-flow nucleophilic pentafluoroethylation using pentafluoroethane (HC2F5, HFC-125), a nontoxic, inexpensive, and commercially available greenhouse gas, is described. The micro-flow pentafluoroethylation by HFC-125 proceeded smoothly at room temperature or at -10 °C in DMF or toluene in the presence of a potassium base, namely, t-BuOK or KHMDS. A broad range of ketones, aldehydes, and chalcones with various substituted benzene rings were successfully converted to the corresponding pentafluoroethyl carbinols instantly with good to high yields.

[Asymptomatic Congenital Left Ventricular Aneurysm Treated with Endocardial Linear Infarct Exclusion Technique( ELIET):Report of a Case].

Surgical repair of asymptomatic congenital left ventricular aneurysm is poorly reported. A 30-yearold man presented with an asymptomatic abnormal electrocardiogram. Computed tomography (CT) and angiography revealed a congenital left ventricular aneurysm, and surgical repair was conducted with endocardial linear infarct exclusion technique (ELIET). His postoperative course was uneventful. Postoperative CT showed an elliptical cardiac shape with no recurrence of aneurysm. ELIET would serve as a surgical procedure for congenital left ventricular aneurysm.

Regulation of Rhcg, an ammonia transporter, by aldosterone in the kidney.

Rhesus C glycoprotein (Rhcg), an ammonia transporter, is a key molecule in urinary acid excretion and is expressed mainly in the intercalated cells (ICs) of the renal collecting duct. In the present study we investigated the role of aldosterone in the regulation of Rhcg expression. In in vivo experiments using C57BL/6J mice, Western blot analysis showed that continuous subcutaneous administration of aldosterone increased the expression of Rhcg in membrane fraction of the kidney. Supplementation of potassium inhibited the effect of aldosterone on the Rhcg. Next, mice were subjected to adrenalectomy with or without administration of aldosterone, and then ad libitum 0.14 M NH4Cl containing water was given. NH4Cl load increased the expression of Rhcg in membrane fraction. Adrenalectomy decreased NH4Cl-induced Rhcg expression, which was restored by administration of aldosterone. Immunohistochemical studies revealed that NH4Cl load induced the localization of Rhcg at the apical membrane of ICs in the outer medullary collecting duct. Adrenalectomy decreased NH4Cl-induced membrane localization of Rhcg, which was restored by administration of aldosterone. For in vitro experiments, IN-IC cells, an immortalized cell line stably expressing Flag-tagged Rhcg (Rhcg-Flag), were used. Western blot analysis showed that aldosterone increased the expression of Rhcg-Flag in membrane fraction, while the increase in extracellular potassium level inhibited the effect of aldosterone. Both spironolactone and GÓ§6983, a PKC inhibitor, inhibited the expression of Rhcg-Flag in the membrane fraction. These results suggest that aldosterone regulates the membrane expression of Rhcg through the mineralocorticoid receptor and PKC pathways, which is modulated by extracellular potassium level.

Pentafluoroethylation of Carbonyl Compounds by HFC-125 via the Encapsulation of the K Cation with Glymes.

A simple protocol to overcome the explosive pentafluoroethylation of carbonyl compounds by HFC-125 is described. The use of potassium (K) bases with triglyme or tetraglyme as a solvent safely yields the pentafluoroethylation products in good to high yields. The experimental results suggest that an encapsulation of the K cation by glymes as K(glyme)2 inhibits the contact between the K cation and the reactive anionic pentafluoroethyl counterion, preventing their transformation into KF and explosive tetrafluoroethylene (TFE). The generation of sterically demanding [K(G3)2]+ and [K(G4)2]+ is an effective way as an unstable pentafluoroethyl anion reservoir.

Synthesis of trifluoromethyl ketones by nucleophilic trifluoromethylation of esters under a fluoroform/KHMDS/triglyme system.

A straightforward method that enables the formation of biologically attractive trifluoromethyl ketones from readily available methyl esters using the potent greenhouse gas fluoroform (HCF3, HFC-23) was developed. The combination of fluoroform and KHMDS in triglyme at -40 °C was effective for this transformation, with good yields as high as 92%. Substrate scope of the trifluoromethylation procedure was explored for aromatic, aliphatic, and conjugated methyl esters. This study presents a straightforward trifluoromethylation process of various methyl esters that convert well to the corresponding trifluoromethyl ketones. The tolerance of various pharmacophores under the reaction conditions was also explored.

Inactivation of a Frameshift TSH Receptor Variant Val711Phefs*18 is Due to Acquisition of a Hydrophobic Degron.

CONTEXT: Inactivating variants of thyrotropin (thyroid-stimulating hormone; TSH) receptor (TSHR) cause congenital hypothyroidism. More than 60 such variants have been reported so far, most of which were located in the extracellular or transmembrane domain. OBJECTIVE: We report the identification and characterization of a frameshift TSHR variant in the intracytoplasmic C-tail region. METHODS: Sequencing of TSHR was performed in a patient with congenital hypothyroidism. The functionality of the identified variants was assessed by expressing TSHR in HEK293 cells and measuring TSH-dependent activation of the cAMP-response element-luciferase reporter. A series of systematic mutagenesis experiments were performed to characterize the frameshifted amino acid sequence. RESULTS: The proband was heterozygous for a known TSHR variant (p.Arg519His) and a novel frameshift TSHR variant (p.Val711Phefs*18), which removed 54 C-terminal residues and added a 17-amino acid frameshifted sequence. The loss of function of Val711Phefs*18-TSHR was confirmed in vitro, but the function of Val711*-TSHR was found to be normal. Western blotting showed the low protein expression of Val711Phefs*18-TSHR. Fusion of the frameshift sequence to green fluorescent protein or luciferase induced inactivation of them, indicating that the sequence acted as a degron. A systematic mutagenesis study revealed that the density of hydrophobic residues in the frameshift sequence determined the stability. Eight additional frameshift TSHR variants that covered all possible shifted frames in C-tail were created, and another frameshift variant (Thr748Profs*27) with similar effect was found. CONCLUSIONS: We characterized a naturally occurring frameshift TSHR variant located in C-tail, and provided a unique evidence that hydrophobicity in the C-terminal region of the receptor affects protein stability.

Impact of Magnesium Stearate Content: Modeling of Drug Degradation Using a Modified Arrhenius Equation.

To accelerate drug development, the pharmaceutical industry is working to shorten and improve studies on stability. The Accelerated Stability Assessment Program (ASAP) incorporating the humidity-corrected Arrhenius equation as an accelerated methodology has been proposed for both drug substances and drug products. In this study, the effect of magnesium stearate (MgSt) content on the chemical stability of acetylsalicylic acid was evaluated as a model system of drug-excipient compatibility studies using ASAP. In the acetylsalicylic acid powder blends, temperature and humidity showed a first-order linear response to the natural logarithm of the reaction rate constant, and MgSt content also showed a first-order linear response. A polynomial model was built in which temperature, humidity, and MgSt content were independent each other. The fitting index of the model, the coefficient of determination, was 0.9567, which was a good fit. In the long-term stability study (25 °C/60% relative humidity, 6 months), there was good agreement in total between measured values and model-predicted values. Using this model, we inferred that the degradation rates were depended on MgSt content at the fixed temperature and humidity because the micro-environmental pH of the excipient was catalytically affected. Applying this model equation can significantly reduce the duration of formulation design and stability studies and save time and costs in drug development.