Loss of Natriuretic Peptide Receptor C Enhances Sinoatrial Node Dysfunction in Aging and Frail Mice.

Heart rate (HR) is controlled by the sinoatrial node (SAN). SAN dysfunction is highly prevalent in aging; however, not all individuals age at the same rate. Rather, health status during aging is affected by frailty. Natriuretic peptides regulate SAN function in part by activating natriuretic peptide receptor C (NPR-C). The impacts of NPR-C on HR and SAN function in aging and as a function of frailty are unknown. Frailty was measured in aging wild-type and NPR-C knockout (NPR-C-/-) mice using a mouse clinical frailty index (FI). HR and SAN structure and function were investigated using intracardiac electrophysiology in anesthetized mice, high-resolution optical mapping in intact atrial preparations, histology, and molecular biology. NPR-C-/- mice rapidly became frail leading to shortened life span. HR was reduced and SAN recovery time was increased in older versus younger mice, and these changes were exacerbated in NPR-C-/- mice; however, there was substantial variability among age groups and genotypes. HR and SAN recovery time were correlated with FI score and fell along a continuum regardless of age or genotype. Optical mapping demonstrates impairments in SAN function that were also correlated with FI score. SAN fibrosis was increased in aged and NPR-C-/- mice and was graded by FI score. Loss of NPR-C results in accelerated aging and rapid decline in health status in association with impairments in HR and SAN function. Frailty assessment was effective and better able to distinguish aging-dependent changes in SAN function in the setting of shortened life span due to loss of NPR-C.

Atrial Fibrillation in Aging and Frail Mice: Modulation by Natriuretic Peptide Receptor C.

[Figure: see text].

The role of digital tools in the delivery of genomic medicine: enhancing patient-centered care.

PURPOSE: Alternative models of genetic counseling are needed to meet the rising demand for genomic sequencing. Digital tools have been proposed as a method to augment traditional counseling and reduce burden on professionals; however, their role in delivery of genetic counseling is not established. This study explored the role of the Genomics ADvISER, a digital decision aid, in delivery of genomic counseling. METHODS: We performed secondary analysis of 52 pretest genetic counseling sessions that were conducted over the course of a randomized controlled trial evaluating the effectiveness of the Genomics ADvISER. As part of the trial, participants were randomized to receive standard counseling or use the tool and then speak with a counselor. A qualitative interpretive description approach using thematic analysis and constant comparison was used for analysis. RESULTS: In the delivery of genomic counseling, the Genomics ADvISER contributed to enhancing counseling by (1) promoting informed dialogue, (2) facilitating preference-sensitive deliberation, and (3) deepening personalization of decisions, all of which represent fundamental principles of patient-centered care: providing clear high-quality information, respecting patients' values, preferences, and expressed needs, and providing emotional support. CONCLUSION: This study demonstrates that our digital tool contributed to enhancing patient-centered care in the delivery of genomic counseling.

Loss of insulin signaling may contribute to atrial fibrillation and atrial electrical remodeling in type 1 diabetes.

Atrial fibrillation (AF) is prevalent in diabetes mellitus (DM); however, the basis for this is unknown. This study investigated AF susceptibility and atrial electrophysiology in type 1 diabetic Akita mice using in vivo intracardiac electrophysiology, high-resolution optical mapping in atrial preparations, and patch clamping in isolated atrial myocytes. qPCR and western blotting were used to assess ion channel expression. Akita mice were highly susceptible to AF in association with increased P-wave duration and slowed atrial conduction velocity. In a second model of type 1 DM, mice treated with streptozotocin (STZ) showed a similar increase in susceptibility to AF. Chronic insulin treatment reduced susceptibility and duration of AF and shortened P-wave duration in Akita mice. Atrial action potential (AP) morphology was altered in Akita mice due to a reduction in upstroke velocity and increases in AP duration. In Akita mice, atrial Na+ current (INa) and repolarizing K+ current (IK) carried by voltage gated K+ (Kv1.5) channels were reduced. The reduction in INa occurred in association with reduced expression of SCN5a and voltage gated Na+ (NaV1.5) channels as well as a shift in INa activation kinetics. Insulin potently and selectively increased INa in Akita mice without affecting IK Chronic insulin treatment increased INa in association with increased expression of NaV1.5. Acute insulin also increased INa, although to a smaller extent, due to enhanced insulin signaling via phosphatidylinositol 3,4,5-triphosphate (PIP3). Our study reveals a critical, selective role for insulin in regulating atrial INa, which impacts susceptibility to AF in type 1 DM.

NPR-C (Natriuretic Peptide Receptor-C) Modulates the Progression of Angiotensin II-Mediated Atrial Fibrillation and Atrial Remodeling in Mice.

BACKGROUND: Atrial fibrillation (AF) commonly occurs in hypertension and in association with elevated Ang II (angiotensin II) levels. The specific mechanisms underlying Ang II-mediated AF are unclear, and interventions to prevent the effects of Ang II are lacking. NPs (natriuretic peptides), which elicit their effects through specific NP receptors, including NPR-C (natriuretic peptide receptor-C), are cardioprotective hormones that affect cardiac structure and function. METHODS: This study used wild-type and NPR-C knockout (NPR-C-/-) mice to investigate the effects of Ang II (3 mg/kg per day for 3 weeks) on AF susceptibility and atrial function using in vivo electrophysiology, high-resolution optical mapping, patch clamping, and molecular biology. In some experiments, wild-type mice were cotreated with Ang II and the NPR-C agonist cANF (0.07-0.14 mg/kg per day) for 3 weeks. RESULTS: In wild-type mice, Ang II increased susceptibility to AF in association with a prolongation of P-wave duration, increased atrial refractory period, and slowed atrial conduction. These effects were exacerbated in Ang II-treated NPR-C-/- mice. Ang II prolonged action potential duration and reduced action potential upstroke velocity (Vmax). These effects were greater in left atrial myocytes from Ang II-treated NPR-C-/- mice. Ang II also increased fibrosis in both atria in wild-type mice, whereas Ang II-treated NPR-C-/- mice exhibited substantially higher fibrosis throughout the atria. Fibrotic responses were associated with changes in expression of profibrotic genes, including TGFß and TIMP1. Cotreating wild-type mice with Ang II and the NPR-C agonist cANF dose dependently reduced AF inducibility by preventing some of the Ang II-induced changes in atrial myocyte electrophysiology and preventing fibrosis throughout the atria. CONCLUSIONS: NPR-C may represent a new target for the prevention of Ang II-induced AF via protective effects on atrial electrical and structural remodeling.

Distinct patterns of atrial electrical and structural remodeling in angiotensin II mediated atrial fibrillation.

Atrial fibrillation (AF) is prevalent in hypertension and elevated angiotensin II (Ang II); however, the mechanisms by which Ang II leads to AF are poorly understood. Here, we investigated the basis for this in mice treated with Ang II or saline for 3 weeks. Ang II treatment increased susceptibility to AF compared to saline controls in association with increases in P wave duration and atrial effective refractory period, as well as reductions in right and left atrial conduction velocity. Patch-clamp studies demonstrate that action potential (AP) duration was prolonged in right atrial myocytes from Ang II treated mice in association with a reduction in repolarizing K+ currents. In contrast, APs in left atrial myocytes from Ang II treated mice showed reductions in upstroke velocity and overshoot, as well as greater prolongations in AP duration. Ang II reduced Na+ current (INa) in the left, but not the right atrium. This reduction in INa was reversible following inhibition of protein kinase C (PKC) and PKCα expression was increased selectively in the left atrium in Ang II treated mice. The transient outward K+ current (Ito) showed larger reductions in the left atrium in association with a shift in the voltage dependence of activation. Finally, Ang II caused fibrosis throughout the atria in association with changes in collagen expression and regulators of the extracellular matrix. This study demonstrates that hypertension and elevated Ang II cause distinct patterns of electrical and structural remodeling in the right and left atria that collectively create a substrate for AF.

A beginner's guide to understanding and implementing the genetic modification of zebrafish.

Zebrafish are a relevant and useful vertebrate model species to study normal- and patho-physiology, including that of the heart, due to conservation of protein-coding genes, organ system organisation and function, and efficient breeding and housing. Their amenability to genetic modification, particularly compared to other vertebrate species, is another great advantage, and is the focus of this review. A vast number of genetically engineered zebrafish lines and methods for their creation exist, but their incorporation into research programs is hindered by the overwhelming amount of technical details. The purpose of this paper is to provide a simplified guide to the fundamental information required by the uninitiated researcher for the thorough understanding, critical evaluation, and effective implementation of genetic approaches in the zebrafish. First, an overview of existing zebrafish lines generated through large scale chemical mutagenesis, retroviral insertional mutagenesis, and gene and enhancer trap screens is presented. Second, descriptions of commonly-used genetic modification methods are provided including Tol2 transposon, TALENs (transcription activator-like effector nucleases), and CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9). Lastly, design features of genetic modification strategies such as promoters, fluorescent reporters, and conditional transgenesis, are summarised. As a comprehensive resource containing both background information and technical notes of how to obtain or generate zebrafish, this review compliments existing resources to facilitate the use of genetically-modified zebrafish by researchers who are new to the field.

Natriuretic Peptide Receptor-C Protects Against Angiotensin II-Mediated Sinoatrial Node Disease in Mice.

Sinoatrial node (SAN) disease mechanisms are poorly understood, and therapeutic options are limited. Natriuretic peptide(s) (NP) are cardioprotective hormones whose effects can be mediated partly by the NP receptor C (NPR-C). We investigated the role of NPR-C in angiotensin II (Ang II)-mediated SAN disease in mice. Ang II caused SAN disease due to impaired electrical activity in SAN myocytes and increased SAN fibrosis. Strikingly, Ang II treatment in NPR-C-/- mice worsened SAN disease, whereas co-treatment of wild-type mice with Ang II and a selective NPR-C agonist (cANF) prevented SAN dysfunction. NPR-C may represent a new target to protect against the development of Ang II-induced SAN disease.

Cardiac Electrophysiological Effects of Light-Activated Chloride Channels.

During the last decade, optogenetics has emerged as a paradigm-shifting technique to monitor and steer the behavior of specific cell types in excitable tissues, including the heart. Activation of cation-conducting channelrhodopsins (ChR) leads to membrane depolarization, allowing one to effectively trigger action potentials (AP) in cardiomyocytes. In contrast, the quest for optogenetic tools for hyperpolarization-induced inhibition of AP generation has remained challenging. The green-light activated ChR from Guillardia theta (GtACR1) mediates Cl--driven photocurrents that have been shown to silence AP generation in different types of neurons. It has been suggested, therefore, to be a suitable tool for inhibition of cardiomyocyte activity. Using single-cell electrophysiological recordings and contraction tracking, as well as intracellular microelectrode recordings and in vivo optical recordings of whole hearts, we find that GtACR1 activation by prolonged illumination arrests cardiac cells in a depolarized state, thus inhibiting re-excitation. In line with this, GtACR1 activation by transient light pulses elicits AP in rabbit isolated cardiomyocytes and in spontaneously beating intact hearts of zebrafish. Our results show that GtACR1 inhibition of AP generation is caused by cell depolarization. While this does not address the need for optogenetic silencing through physiological means (i.e., hyperpolarization), GtACR1 is a potentially attractive tool for activating cardiomyocytes by transient light-induced depolarization.

Altered heart rate regulation by the autonomic nervous system in mice lacking natriuretic peptide receptor C (NPR-C).

Natriuretic peptides (NPs) play essential roles in the regulation of cardiovascular function. NP effects are mediated by receptors known as NPR-A, NPR-B or NPR-C. NPs have potent effects on regulation of heart rate (HR) by the autonomic nervous system (ANS), but the role of NPR-C in these effects has not been investigated. Accordingly, we have used telemetric ECG recordings in awake, freely moving wildtype and NPR-C knockout (NPR-C-/-) mice and performed heart rate variability (HRV) analysis to assess alterations in sympatho-vagal balance on the heart following loss of NPR-C. Our novel data demonstrate that NPR-C-/- mice are characterized by elevations in HR, reductions in circadian changes in HR and enhanced occurrence of sinus pauses, indicating increased arrhythmogenesis and a loss of HRV. Time domain and frequency domain analyses further demonstrate that HRV is reduced in NPR-C-/- mice in association with a reduction in parasympathetic activity. Importantly, the low frequency to high frequency ratio was increased in NPR-C-/- mice indicating that sympathetic activity is also enhanced. These changes in autonomic regulation were confirmed using atropine and propranolol to antagonize the ANS. These findings illustrate that loss of NPR-C reduces HRV due to perturbations in the regulation of the heart by the ANS.

Atrial structure, function and arrhythmogenesis in aged and frail mice.

Atrial fibrillation (AF) is prevalent in aging populations; however not all individuals age at the same rate. Instead, individuals of the same chronological age can vary in health status from fit to frail. Our objective was to determine the impacts of age and frailty on atrial function and arrhythmogenesis in mice using a frailty index (FI). Aged mice were more frail and demonstrated longer lasting AF compared to young mice. Consistent with this, aged mice showed longer P wave duration and PR intervals; however, both parameters showed substantial variability suggesting differences in health status among mice of similar chronological age. In agreement with this, P wave duration and PR interval were highly correlated with FI score. High resolution optical mapping of the atria demonstrated reduced conduction velocity and action potential duration in aged hearts that were also graded by FI score. Furthermore, aged mice had increased interstitial fibrosis along with changes in regulators of extracellular matrix remodelling, which also correlated with frailty. These experiments demonstrate that aging results in changes in atrial structure and function that create a substrate for atrial arrhythmias. Importantly, these changes were heterogeneous due to differences in health status, which could be identified using an FI.

The impacts of age and frailty on heart rate and sinoatrial node function.

KEY POINTS: Sinoatrial node (SAN) function declines with age; however, not all individuals age at the same rate and health status can vary from fit to frail. Frailty was quantified in young and aged mice using a non-invasive frailty index so that the impacts of age and frailty on heart rate and SAN function could be assessed. SAN function was impaired in aged mice due to alterations in electrical conduction, changes in SAN action potential morphology and fibrosis in the SAN. Changes in SAN function, electrical conduction, action potential morphology and fibrosis were correlated with, and graded by, frailty. This study shows that mice of the same chronological age have quantifiable differences in health status that impact heart rate and SAN function and that these differences in health status can be identified using our frailty index. ABSTRACT: Sinoatrial node (SAN) dysfunction increases with age, although not all older adults are affected in the same way. This is because people age at different rates and individuals of the same chronological age vary in health status from very fit to very frail. Our objective was to determine the impacts of age and frailty on heart rate (HR) and SAN function using a new model of frailty in ageing mice. Frailty, which was quantified in young and aged mice using a frailty index (FI), was greater in aged vs. young mice. Intracardiac electrophysiology demonstrated that HR was reduced whereas SAN recovery time (SNRT) was prolonged in aged mice; however, both parameters showed heteroscedasticity suggesting differences in health status among mice of similar chronological age. Consistent with this, HR and corrected SNRT were correlated with, and graded by, FI score. Optical mapping of the SAN demonstrated that conduction velocity (CV) was reduced in aged hearts in association with reductions in diastolic depolarization (DD) slope and action potential (AP) duration. In agreement with in vivo results, SAN CV, DD slope and AP durations all correlated with FI score. Finally, SAN dysfunction in aged mice was associated with increased interstitial fibrosis and alterations in expression of matrix metalloproteinases, which also correlated with frailty. These findings demonstrate that age-related SAN dysfunction occurs in association with electrical and structural remodelling and that frailty is a critical determinant of health status of similarly aged animals that correlates with changes in HR and SAN function.

Impaired sinoatrial node function and increased susceptibility to atrial fibrillation in mice lacking natriuretic peptide receptor C.

Natriuretic peptides (NPs) are critical regulators of the cardiovascular system that are currently viewed as possible therapeutic targets for the treatment of heart disease. Recent work demonstrates potent NP effects on cardiac electrophysiology, including in the sinoatrial node (SAN) and atria. NPs elicit their effects via three NP receptors (NPR-A, NPR-B and NPR-C). Among these receptors, NPR-C is poorly understood. Accordingly, the goal of this study was to determine the effects of NPR-C ablation on cardiac structure and arrhythmogenesis. Cardiac structure and function were assessed in wild-type (NPR-C(+/+)) and NPR-C knockout (NPR-C(-/-)) mice using echocardiography, intracardiac programmed stimulation, patch clamping, high-resolution optical mapping, quantitative polymerase chain reaction and histology. These studies demonstrate that NPR-C(-/-) mice display SAN dysfunction, as indicated by a prolongation (30%) of corrected SAN recovery time, as well as an increased susceptibility to atrial fibrillation (6% in NPR-C(+/+) vs. 47% in NPR-C(-/-)). There were no differences in SAN or atrial action potential morphology in NPR-C(-/-) mice; however, increased atrial arrhythmogenesis in NPR-C(-/-) mice was associated with reductions in SAN (20%) and atrial (15%) conduction velocity, as well as increases in expression and deposition of collagen in the atrial myocardium. No differences were seen in ventricular arrhythmogenesis or fibrosis in NPR-C(-/-) mice. This study demonstrates that loss of NPR-C results in SAN dysfunction and increased susceptibility to atrial arrhythmias in association with structural remodelling and fibrosis in the atrial myocardium. These findings indicate a critical protective role for NPR-C in the heart.

VIP-dependent increase in F508del-CFTR membrane localization is mediated by PKCε.

The most common cystic fibrosis causing mutation F508del induces early degradation and reduced trafficking of cystic fibrosis transmembrane conductance regulator (CFTR) chloride channels to the apical membrane of epithelial cells. In the human nasal epithelial cells JME/CF15, we previously reported that vasoactive intestinal peptide (VIP) exposure corrects trafficking and membrane insertion of functional F508del-CFTR channels at 37°C. Correction of trafficking was PKA dependent, whereas enhanced membrane localization involved PKC. In the present study, we have identified PKCε as the isoform involved in VIP-dependent F508del-CFTR membrane insertion. Iodide effluxes were used to monitor the presence of VIP-rescued functional F508del-CFTR channels at the surface of JME/CF15 cells maintained at 37°C. Iodide efflux peaks measured in response to stimulation with forskolin were insensitive to PKC α, ß, γ, δ, ζ inhibitors. In contrast, efflux peaks were completely inhibited by pretreatment with the PKCε inhibitor peptide EAVSLKPT with an IC(50) of 4.9 µM or by PKCε small interfering RNA (siRNA). Immunostaining and confocal microscopy confirmed that membrane localization of F508del-CFTR induced by VIP was abolished in the presence of EAVSLKPT but not with other isoform inhibitors. In recombinant baby hamster kidney cells, endogenously expressing PKCε but no VIP receptor, wild-type, and F508del-CFTR sensitivity to cpt-cAMP stimulation was increased by PMA treatment. Biotinylation assays and immunoblots confirmed that PMA (0.5-2 h) induced a greater than threefold increase in membrane CFTR, whereas forskolin had no effect. The PMA effect was abolished by specifically inhibiting PKCε (EAVSLKPT IC(50) = 5.7 µM) but not other PKC isoforms. Taken together, these results indicate that stimulating PKCε by VIP or PMA increases membrane insertion and activity of WT- and F508del-CFTR.

Rescue of functional F508del cystic fibrosis transmembrane conductance regulator by vasoactive intestinal peptide in the human nasal epithelial cell line JME/CF15.

F508del is the most common cystic fibrosis-causing mutation that induces early degradation and poor trafficking of cystic fibrosis transmembrane conductance regulator (CFTR) chloride channels to the apical membrane of epithelial cells. Our previous work in bronchial serous cells showed that vasoactive intestinal peptide (VIP) stimulation of the VPAC(1) receptor enhances CFTR-dependent chloride secretion by increasing its membrane insertion by a protein kinase C (PKC)-dependent pathway. In the present study, we investigated the effect of VIP on F508del-CFTR activity and membrane insertion in the human nasal epithelial cell line JME/CF15, which also expresses the VPAC(1) receptor. At reduced temperature (27 degrees C), which rescues F508del-CFTR trafficking, acute stimulation by VIP of rescued F508del-CFTR channels was protein kinase A (PKA)- and PKC-dependent. One hour of treatment with VIP strongly increased F508del-CFTR activity, with iodide efflux peaks three times higher than with untreated cells. At 37 degrees C, VIP-treated cells, but not untreated controls, showed significant iodide efflux peaks that were sensitive to the CFTR inhibitor 3-[(3-trifluoromethyl)phenyl]-5-[(4-carboxyphenyl)methylene]-2-thioxo-4-thiazolidinone (CFTR(inh)-172). Immunostaining, biotinylation assays, and Western blots confirmed a VIP-induced maturation and membrane insertion of F508del-CFTR at 37 degrees C. The corrector effect of VIP was abolished by the PKA inhibitor N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamidedihydrochloride (H89), whereas Galpha(s) stimulation by cholera toxin significantly increased F508del-CFTR trafficking. On the other hand, membrane localization, but not maturation, of F508del-CFTR was significantly reduced by the PKC inhibitor bisindolylmaleimide X and the G(i/o) protein inhibitor pertussis toxin. VIP treatment had no effect on intracellular calcium or proteasome activity. These results indicate that, in human nasal cells, VIP rescues trafficking and membrane insertion of functional F508del-CFTR channels at physiological temperature by stimulating both PKA- and PKC-dependent pathways.

A comparison of twice- versus once-weekly supervision during pulmonary rehabilitation in chronic obstructive pulmonary disease.

OBJECTIVE: To compare the effects of twice- versus once-weekly supervised pulmonary rehabilitation on exercise capacity and quality of life in patients with chronic obstructive pulmonary disease (COPD). DESIGN: Prospective, randomized, parallel-group study. SETTING: Hospital outpatient physiotherapy department. PARTICIPANTS: Patients with COPD. INTERVENTION: Group 1 (n=46) received 1 supervised exercise session a week and 2 unsupervised sessions; group 2 (n= 45) received 2 supervised exercise sessions a week and 1 unsupervised session for 6 weeks. Patients were assessed at baseline and at 6 weeks, 2 months, and 6 months. MAIN OUTCOME MEASURES: Incremental (ISWT) and endurance (ESWT) shuttle walk tests and Chronic Respiratory Disease Questionnaire (CRDQ). RESULTS: Sixty-six of 91 patients (group 1, n=34; group 2, n=32) completed the 6-week program. There was no significant difference in key outcome measures between the 2 groups (ISWT, 13.50m; 95% confidence interval [CI], -10.06 to 37.15m; ESWT, 72.64s; 95% CI, -96.01 to 241.29s; CRDQ total score, 2.54; 95% CI, -3.16 to 8.24). The results of the ESWTs suggest there may be an interaction between baseline exercise capacity and benefit of pulmonary rehabilitation, with more disabled patients achieving greater benefit if they are supervised twice weekly. Irrespective of group, allocation benefits after pulmonary rehabilitation had almost dissipated by 6 months. CONCLUSIONS: There was no difference in the effectiveness of twice- versus once-weekly supervised pulmonary rehabilitation. This study highlights the need for development of strategies that will maintain the improvement achieved by the initial pulmonary rehabilitation program.