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.

New role for cardiomyocyte Bmal1 in the regulation of sex-specific heart transcriptomes.

It has been well established that cardiovascular diseases exhibit significant differences between sexes in both preclinical models and humans. In addition, there is growing recognition that disrupted circadian rhythms can contribute to the onset and progression of cardiovascular diseases. However little is known about sex differences between the cardiac circadian clock and circadian transcriptomes in mice. Here, we show that the the core clock genes are expressed in common in both sexes but the circadian transcriptome of the mouse heart is very sex-specific. Hearts from female mice expressed significantly more rhythmically expressed genes (REGs) than male hearts and the temporal pattern of REGs was distinctly different between sexes. We next used a cardiomyocyte-specific knock out of the core clock gene, Bmal1, to investigate its role in sex-specific gene expression in the heart. All sex differences in the circadian transcriptomes were significantly diminished with cardiomyocyte-specific loss of Bmal1. Surprisingly, loss of cardiomyocyte Bmal1 also resulted in a roughly 8-fold reduction in the number of all the differentially expressed genes between male and female hearts. We conclude that cardiomyocyte-specific Bmal1, and potentially the core clock mechanism, is vital in conferring sex-specific gene expression in the adult mouse heart.

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.

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.

Detecting organelle-specific activity of potassium channels with a DNA nanodevice.

Cell surface potassium ion (K+) channels regulate nutrient transport, cell migration and intercellular communication by controlling K+ permeability and are thought to be active only at the plasma membrane. Although these channels transit the trans-Golgi network, early and recycling endosomes, whether they are active in these organelles is unknown. Here we describe a pH-correctable, ratiometric reporter for K+ called pHlicKer, use it to probe the compartment-specific activity of a prototypical voltage-gated K+ channel, Kv11.1, and show that this cell surface channel is active in organelles. Lumenal K+ in organelles increased in cells expressing wild-type Kv11.1 channels but not after treatment with current blockers. Mutant Kv11.1 channels, with impaired transport function, failed to increase K+ levels in recycling endosomes, an effect rescued by pharmacological correction. By providing a way to map the organelle-specific activity of K+ channels, pHlicKer technology could help identify new organellar K+ channels or channel modulators with nuanced functions.

Prediction of Kv11.1 potassium channel PAS-domain variants trafficking via machine learning.

Congenital long QT syndrome (LQTS) is characterized by a prolonged QT-interval on an electrocardiogram (ECG). An abnormal prolongation in the QT-interval increases the risk for fatal arrhythmias. Genetic variants in several different cardiac ion channel genes, including KCNH2, are known to cause LQTS. Here, we evaluated whether structure-based molecular dynamics (MD) simulations and machine learning (ML) could improve the identification of missense variants in LQTS-linked genes. To do this, we investigated KCNH2 missense variants in the Kv11.1 channel protein shown to have wild type (WT) like or class II (trafficking-deficient) phenotypes in vitro. We focused on KCNH2 missense variants that disrupt normal Kv11.1 channel protein trafficking, as it is the most common phenotype for LQTS-associated variants. Specifically, we used computational techniques to correlate structural and dynamic changes in the Kv11.1 channel protein PAS domain (PASD) with Kv11.1 channel protein trafficking phenotypes. These simulations unveiled several molecular features, including the numbers of hydrating waters and hydrogen bonding pairs, as well as folding free energy scores, that are predictive of trafficking. We then used statistical and machine learning (ML) (Decision tree (DT), Random forest (RF), and Support vector machine (SVM)) techniques to classify variants using these simulation-derived features. Together with bioinformatics data, such as sequence conservation and folding energies, we were able to predict with reasonable accuracy (≈75%) which KCNH2 variants do not traffic normally. We conclude that structure-based simulations of KCNH2 variants localized to the Kv11.1 channel PASD led to an improvement in classification accuracy. Therefore, this approach should be considered to complement the classification of variant of unknown significance (VUS) in the Kv11.1 channel PASD.

New Insights into Cardiac Ion Channel Regulation 2.0.

Sudden cardiac death (SCD) and arrhythmias represent a global public health problem, accounting for 15-20% of all deaths [...].

Defining the age-dependent and tissue-specific circadian transcriptome in male mice.

Cellular circadian clocks direct a daily transcriptional program that supports homeostasis and resilience. Emerging evidence has demonstrated age-associated changes in circadian functions. To define age-dependent changes at the systems level, we profile the circadian transcriptome in the hypothalamus, lung, heart, kidney, skeletal muscle, and adrenal gland in three age groups. We find age-dependent and tissue-specific clock output changes. Aging reduces the number of rhythmically expressed genes (REGs), indicative of weakened circadian control. REGs are enriched for the hallmarks of aging, adding another dimension to our understanding of aging. Analyzing differential gene expression within a tissue at four different times of day identifies distinct clusters of differentially expressed genes (DEGs). Increased variability of gene expression across the day is a common feature of aged tissues. This analysis extends the landscape for understanding aging and highlights the impact of aging on circadian clock function and temporal changes in gene expression.

Guidelines for assessment of cardiac electrophysiology and arrhythmias in small animals.

Cardiac arrhythmias are a major cause of morbidity and mortality worldwide. Although recent advances in cell-based models, including human-induced pluripotent stem cell-derived cardiomyocytes (iPSC-CM), are contributing to our understanding of electrophysiology and arrhythmia mechanisms, preclinical animal studies of cardiovascular disease remain a mainstay. Over the past several decades, animal models of cardiovascular disease have advanced our understanding of pathological remodeling, arrhythmia mechanisms, and drug effects and have led to major improvements in pacing and defibrillation therapies. There exist a variety of methodological approaches for the assessment of cardiac electrophysiology and a plethora of parameters may be assessed with each approach. This guidelines article will provide an overview of the strengths and limitations of several common techniques used to assess electrophysiology and arrhythmia mechanisms at the whole animal, whole heart, and tissue level with a focus on small animal models. We also define key electrophysiological parameters that should be assessed, along with their physiological underpinnings, and the best methods with which to assess these parameters.

Mutation-Specific Differences in Kv7.1 (KCNQ1) and Kv11.1 (KCNH2) Channel Dysfunction and Long QT Syndrome Phenotypes.

The electrocardiogram (ECG) empowered clinician scientists to measure the electrical activity of the heart noninvasively to identify arrhythmias and heart disease. Shortly after the standardization of the 12-lead ECG for the diagnosis of heart disease, several families with autosomal recessive (Jervell and Lange-Nielsen Syndrome) and dominant (Romano-Ward Syndrome) forms of long QT syndrome (LQTS) were identified. An abnormally long heart rate-corrected QT-interval was established as a biomarker for the risk of sudden cardiac death. Since then, the International LQTS Registry was established; a phenotypic scoring system to identify LQTS patients was developed; the major genes that associate with typical forms of LQTS were identified; and guidelines for the successful management of patients advanced. In this review, we discuss the molecular and cellular mechanisms for LQTS associated with missense variants in KCNQ1 (LQT1) and KCNH2 (LQT2). We move beyond the "benign" to a "pathogenic" binary classification scheme for different KCNQ1 and KCNH2 missense variants and discuss gene- and mutation-specific differences in K+ channel dysfunction, which can predispose people to distinct clinical phenotypes (e.g., concealed, pleiotropic, severe, etc.). We conclude by discussing the emerging computational structural modeling strategies that will distinguish between dysfunctional subtypes of KCNQ1 and KCNH2 variants, with the goal of realizing a layered precision medicine approach focused on individuals.

How Functional Genomics Can Keep Pace With VUS Identification.

Over the last two decades, an exponentially expanding number of genetic variants have been identified associated with inherited cardiac conditions. These tremendous gains also present challenges in deciphering the clinical relevance of unclassified variants or variants of uncertain significance (VUS). This review provides an overview of the advancements (and challenges) in functional and computational approaches to characterize variants and help keep pace with VUS identification related to inherited heart diseases.

Time Restricted Feeding to the Light Cycle Dissociates Canonical Circadian Clocks and Physiological Rhythms in Heart Rate.

Circadian rhythms are approximate 24-h biological cycles that optimize molecular and physiological functions to predictable daily environmental changes in order to maintain internal and organismal homeostasis. Environmental stimuli (light, feeding, activity) capable of altering the phase of molecular rhythms are important tools employed by circadian biologists to increase understanding of the synchronization of circadian rhythms to the environment and to each other within multicellular systems. The central circadian clock, located in the suprachiasmatic nucleus (SCN) of the hypothalamus is largely responsive to light and is thought to entrain the phase of peripheral clocks via neurohumoral signals. Mice are nocturnal and consume most of their food during the dark cycle. Early studies demonstrated that altered metabolic cues in the form of time restricted feeding, specifically, feeding mice during the light cycle, resulted in an uncoupling of molecular clocks in peripheral tissues with those from the SCN. These studies showed as much as a 12-h shift in gene expression in some peripheral tissues but not others. The shifts occurred without corresponding changes in the central clock in the brain. More recent studies have demonstrated that changes in cardiac physiology (heart rate, MAP) in response to time of food intake occur independent of the cardiac molecular clock. Understanding differences in the physiology/function and gene expression in other organs both independently and in relation to the heart in response to altered feeding will be important in dissecting the roles of the various clocks throughout the body, as well as, understanding their links to cardiovascular pathology.

Research Opportunities in Autonomic Neural Mechanisms of Cardiopulmonary Regulation: A Report From the National Heart, Lung, and Blood Institute and the National Institutes of Health Office of the Director Workshop.

This virtual workshop was convened by the National Heart, Lung, and Blood Institute, in partnership with the Office of Strategic Coordination of the Office of the National Institutes of Health Director, and held September 2 to 3, 2020. The intent was to assemble a multidisciplinary group of experts in basic, translational, and clinical research in neuroscience and cardiopulmonary disorders to identify knowledge gaps, guide future research efforts, and foster multidisciplinary collaborations pertaining to autonomic neural mechanisms of cardiopulmonary regulation. The group critically evaluated the current state of knowledge of the roles that the autonomic nervous system plays in regulation of cardiopulmonary function in health and in pathophysiology of arrhythmias, heart failure, sleep and circadian dysfunction, and breathing disorders. Opportunities to leverage the Common Fund's SPARC (Stimulating Peripheral Activity to Relieve Conditions) program were characterized as related to nonpharmacologic neuromodulation and device-based therapies. Common themes discussed include knowledge gaps, research priorities, and approaches to develop novel predictive markers of autonomic dysfunction. Approaches to precisely target neural pathophysiological mechanisms to herald new therapies for arrhythmias, heart failure, sleep and circadian rhythm physiology, and breathing disorders were also detailed.

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.

A High-Throughput Screening Assay to Identify Drugs that Can Treat Long QT Syndrome Caused by Trafficking-Deficient KV11.1 (hERG) Variants.

Loss-of-function (LOF) variants in the KV11.1 potassium channel cause long QT syndrome (LQTS). Most variants disrupt intracellular channel transport (trafficking) to the cell membrane. Since some channel inhibitors improve trafficking of KV11.1 variants, a high-throughput screening (HTS) assay to detect trafficking enhancement would be valuable to the identification of drug candidates. The thallium (Tl+) flux assay technique, widely used for drug screening, was optimized using human embryonic kidney (HEK-293) cells expressing a trafficking-deficient KV11.1 variant in 384-well plates. Assay quality was assessed using Z prime (Z') scores comparing vehicle to E-4031, a drug that increases KV11.1 membrane trafficking. The optimized assay was validated by immunoblot, electrophysiology experiments, and a pilot drug screen. The combination of: 1) truncating the trafficking-deficient variant KV11.1-G601S (KV11.1-G601S-G965*X) with the addition of 2) KV11.1 channel activator (VU0405601) and 3) cesium (Cs+) to the Tl+ flux assay buffer resulted in an outstanding Z' of 0.83. To validate the optimized trafficking assay, we carried out a pilot screen that identified three drugs (ibutilide, azaperone, and azelastine) that increase KV11.1 trafficking. The new assay exhibited 100% sensitivity and specificity. Immunoblot and voltage-clamp experiments confirmed that all three drugs identified by the new assay improved membrane trafficking of two additional LQTS KV11.1 variants. We report two new ways to increase target-specific activity in trafficking assays-genetic modification and channel activation-that yielded a novel HTS assay for identifying drugs that improve membrane expression of pathogenic KV11.1 variants. SIGNIFICANCE STATEMENT: This manuscript reports the development of a high-throughput assay (thallium flux) to identify drugs that can increase function in KV11.1 variants that are trafficking-deficient. Two key aspects that improved the resolving power of the assay and could be transferable to other ion channel trafficking-related assays include genetic modification and channel activation.

Effects of opioid receptor agonist and antagonist medications on electrocardiogram changes and presentation of cardiac arrhythmia: review article.

BACKGROUND/PURPOSE: Mortality associated with prescription opioids has significantly increased over the past few decades and is considered a global pandemic. Prescribed opioids can cause cardiac arrhythmias, leading to fatal outcomes and unexpected death, even in the absence of structural cardiac disease. Despite the extent of cardiac toxicity and death associated with these medications, there is limited data to suggest their influences on cardiac electrophysiology and arrhythmias, with the exception of methadone. The goal of our review is to describe the possible mechanisms and to review the different ECG changes and arrhythmias that have been reported. METHODS: A literature search was performed using Google Scholar, PubMed, Springer, Ovid, and Science Direct to identify studies that demonstrated the use of prescription opioids leading to electrocardiogram (ECG) changes and cardiac arrhythmias. RESULTS: Many of the commonly prescribed opioid medications can uniquely effect the ECG, and can lead to the development of various cardiac arrhythmias. One of the most significant side effects of these drugs is QTc interval prolongation, especially when administered to patients with a baseline risk for QTc prolongation. A prolonged QTc interval can cause lethal torsades de pointes and ventricular fibrillation. Obtaining an ECG at baseline, following a dosage increase, or after switching an opioid medication, is appropriate in patients taking certain prescribed opioids. Opioids are often used first line for the treatment of acute and chronic pain, procedural sedation, medication opioid use disorders, and maintenance therapy. CONCLUSIONS: To reduce the risk of cardiac arrhythmias and to improve patient outcomes, consideration of accurate patient selection, concomitant medications, electrolyte monitoring, and vigilant ECG monitoring should be considered.

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.