Natriuretic Peptide Receptor B Protects Against Atrial Fibrillation by Controlling Atrial cAMP Via Phosphodiesterase 2.

BACKGROUND: ß-AR (ß-adrenergic receptor) stimulation regulates atrial electrophysiology and Ca2+ homeostasis via cAMP-dependent mechanisms; however, enhanced ß-AR signaling can promote atrial fibrillation (AF). CNP (C-type natriuretic peptide) can also regulate atrial electrophysiology through the activation of NPR-B (natriuretic peptide receptor B) and cGMP-dependent signaling. Nevertheless, the role of NPR-B in regulating atrial electrophysiology, Ca2+ homeostasis, and atrial arrhythmogenesis is incompletely understood. METHODS: Studies were performed using atrial samples from human patients with AF or sinus rhythm and in wild-type and NPR-B-deficient (NPR-B+/-) mice. Studies were conducted in anesthetized mice by intracardiac electrophysiology, in isolated mouse atrial preparations using high-resolution optical mapping, in isolated mouse and human atrial myocytes using patch-clamping and Ca2+ imaging, and in mouse and human atrial tissues using molecular biology. RESULTS: Atrial NPR-B protein levels were reduced in patients with AF, and NPR-B+/- mice were more susceptible to AF. Atrial cGMP levels and PDE2 (phosphodiesterase 2) activity were reduced in NPR-B+/- mice leading to larger increases in atrial cAMP in the presence of the ß-AR agonist isoproterenol. NPR-B+/- mice displayed larger increases in action potential duration and L-type Ca2+ current in the presence of isoproterenol. This resulted in the occurrence of spontaneous sarcoplasmic reticulum Ca2+ release events and delayed afterdepolarizations in NPR-B+/- atrial myocytes. Phosphorylation of the RyR2 (ryanodine receptor) and phospholamban was increased in NPR-B+/- atria in the presence of isoproterenol compared with the wildtypes. C-type natriuretic peptide inhibited isoproterenol-stimulated L-type Ca2+ current through PDE2 in mouse and human atrial myocytes. CONCLUSIONS: NPR-B protects against AF by preventing enhanced atrial responses to ß-adrenergic receptor agonists.

Genetic Atrial Cardiomyopathies: Common Features, Specific Differences, and Broader Relevance to Understanding Atrial Cardiomyopathy.

Atrial cardiomyopathy is a condition that causes electrical and contractile dysfunction of the atria, often along with structural and functional changes. Atrial cardiomyopathy most commonly occurs in conjunction with ventricular dysfunction, in which case it is difficult to discern the atrial features that are secondary to ventricular dysfunction from those that arise as a result of primary atrial abnormalities. Isolated atrial cardiomyopathy (atrial-selective cardiomyopathy [ASCM], with minimal or no ventricular function disturbance) is relatively uncommon and has most frequently been reported in association with deleterious rare genetic variants. The genes involved can affect proteins responsible for various biological functions, not necessarily limited to the heart but also involving extracardiac tissues. Atrial enlargement and atrial fibrillation are common complications of ASCM and are often the predominant clinical features. Despite progress in identifying disease-causing rare variants, an overarching understanding and approach to the molecular pathogenesis, phenotypic spectrum, and treatment of genetic ASCM is still lacking. In this review, we aim to analyze the literature relevant to genetic ASCM to understand the key features of this rather rare condition, as well as to identify distinct characteristics of ASCM and its arrhythmic complications that are related to specific genotypes. We outline the insights that have been gained using basic research models of genetic ASCM in vitro and in vivo and correlate these with patient outcomes. Finally, we provide suggestions for the future investigation of patients with genetic ASCM and improvements to basic scientific models and systems. Overall, a better understanding of the genetic underpinnings of ASCM will not only provide a better understanding of this condition but also promises to clarify our appreciation of the more commonly occurring forms of atrial cardiomyopathy associated with ventricular dysfunction.

Regional and temporal progression of atrial remodeling in angiotensin II mediated atrial fibrillation.

Atrial fibrillation (AF) is associated with electrical and structural remodeling in the atria; however, the regional and temporal progression of atrial remodeling is incompletely understood. The objective of this study was to investigate the regional and temporal progression of atrial remodeling leading to changes in AF susceptibility in angiotensin II (Ang II) mediated hypertension. Mice were infused with Ang II for 3, 10 or 21 days. AF susceptibility and atrial electrophysiology were studied in vivo using intracardiac electrophysiology. Right and left atrial myocyte electrophysiology was studied using patch-clamping. Atrial fibrosis was assessed histologically. P wave duration and atrial effective refractory period increased progressively from 3 to 21 days of Ang II. AF susceptibility tended to be increased at 10 days of Ang II and was elevated at 21 days of Ang II. Left, but not right, atrial AP upstroke velocity and Na+ current were reduced at 10 and 21 days of Ang II. Left atrial action potential (AP) duration increased progressively from 3 to 21 days of Ang II due to reductions in repolarizing K+ current. Right atrial AP prolongation was increased only after 21 days of Ang II. Left and right atrial fibrosis developed progressively from 3 to 21 days, but increases were larger in the left atrium. In conclusion, Ang II mediated atrial electrical and structural remodeling develop earlier and more extensively in the left atrium compared to the right atrium, providing insight into how atrial remodeling leads to enhanced AF susceptibility in Ang II mediated hypertension.

Natriuretic peptide receptor B maintains heart rate and sinoatrial node function via cyclic GMP-mediated signalling.

AIMS: Heart rate (HR) is a critical indicator of cardiac performance that is determined by sinoatrial node (SAN) function and regulation. Natriuretic peptides, including C-type NP (CNP), have been shown to modulate ion channel function in the SAN when applied exogenously. CNP is the only NP that acts as a ligand for natriuretic peptide receptor-B (NPR-B). Despite these properties, the ability of CNP and NPR-B to regulate HR and intrinsic SAN automaticity in vivo, and the mechanisms by which it does so, are incompletely understood. Thus, the objective of this study was to determine the role of NPR-B signalling in regulating HR and SAN function. METHODS AND RESULTS: We have used NPR-B deficient mice (NPR-B+/-) to study HR regulation and SAN function using telemetry in conscious mice, intracardiac electrophysiology in anaesthetized mice, high-resolution optical mapping in isolated SAN preparations, patch-clamping in isolated SAN myocytes, and molecular biology in isolated SAN tissue. These studies demonstrate that NPR-B+/- mice exhibit slow HR, increased corrected SAN recovery time, and slowed SAN conduction. Spontaneous AP firing frequency in isolated SAN myocytes was impaired in NPR-B+/- mice due to reductions in the hyperpolarization activated current (If) and L-type Ca2+ current (ICa,L). If and ICa,L were reduced due to lower cGMP levels and increased hydrolysis of cAMP by phosphodiesterase 3 (PDE3) in the SAN. Inhibiting PDE3 or restoring cGMP signalling via application of 8-Br-cGMP abolished the reductions in cAMP, AP firing, If, and ICa,L, and normalized SAN conduction, in the SAN in NPR-B+/- mice. NPR-B+/- mice did not exhibit changes in SAN fibrosis and showed no evidence of cardiac hypertrophy or changes in ventricular function. CONCLUSIONS: NPR-B plays an essential physiological role in maintaining normal HR and SAN function by modulating ion channel function in SAN myocytes via a cGMP/PDE3/cAMP signalling mechanism.

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.

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.

The impact of ovariectomy on cardiac excitation-contraction coupling is mediated through cAMP/PKA-dependent mechanisms.

Ovariectomy (OVX) promotes sarcoplasmic reticulum (SR) Ca2+ overload in ventricular myocytes. We hypothesized that the cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA) pathway contributes to this Ca2+ dysregulation. Myocytes were isolated from adult female C57BL/6 mice following either OVX or sham surgery (surgery at ≈1mos). Contractions, Ca2+ concentrations (fura-2) and ionic currents were measured simultaneously (37°C, 2Hz) in voltage-clamped myocytes. Intracellular cAMP levels were determined with an enzyme immunoassay; phosphodiesterase (PDE) and adenylyl cyclase (AC) isoform expression was examined with qPCR. Ca2+ currents were similar in myocytes from sham and OVX mice but Ca2+ transients, excitation-contraction (EC)-coupling gain, SR content and contractions were larger in OVX than sham cells. To determine if the cAMP/PKA pathway mediated OVX-induced alterations in EC-coupling, cardiomyocytes were incubated with the PKA inhibitor H-89 (2µM), which abolished baseline differences. While basal intracellular cAMP did not differ, levels were higher in OVX than sham in the presence of a non-selective PDE inhibitor (300µM IBMX), or an AC activator (10µM forskolin). This suggests the production of cAMP by AC and its breakdown by PDE were enhanced by OVX. Consistent with this, mRNA levels for both AC5 and PDE4A were higher in OVX in comparison to sham. Differences in Ca2+ homeostasis and contractions were abolished when sham and OVX cells were dialyzed with patch pipettes containing the same concentration of 8-bromoadenosine-cAMP (50µM). Interestingly, selective inhibition of PDE4 increased Ca2+ current only in OVX cells. Together, these findings suggest that estrogen suppresses SR Ca2+ release and that this is regulated, at least in part, by the cAMP/PKA pathway. These changes in the cAMP/PKA pathway may promote Ca2+ dysregulation and cardiovascular disease when ovarian estrogen levels fall. These results advance our understanding of female-specific cardiomyocyte mechanisms that may affect responses to therapeutic interventions in older women.

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.

Altered parasympathetic nervous system regulation of the sinoatrial node in Akita diabetic mice.

Cardiovascular autonomic neuropathy (CAN) is a serious complication of diabetes mellitus that impairs autonomic regulation of heart rate (HR). This has been attributed to damage to the nerves that modulate spontaneous pacemaker activity in the sinoatrial node (SAN). Our objective was to test the hypothesis that impaired parasympathetic regulation of HR in diabetes is due to reduced responsiveness of the SAN to parasympathetic agonists. We used the Akita mouse model of type 1 diabetes to study the effects of the parasympathetic agonist carbachol (CCh) on SAN function using intracardiac programmed stimulation, high resolution optical mapping and patch-clamping of SAN myocytes. CCh decreased HR by 30% and increased corrected SAN recovery time (cSNRT) by 123% in wildtype mice. In contrast, CCh only decreased HR by 12%, and only increased cSNRT by 37% in Akita mice. These alterations were due to smaller effects of CCh on SAN electrical conduction and spontaneous action potential firing in isolated SAN myocytes. Voltage clamp experiments demonstrate that the acetylcholine-activated K(+) current (IKACh) is reduced in Akita SAN myocytes due to enhanced desensitization and faster deactivation kinetics. These IKACh alterations were normalized by treating Akita SAN myocytes with PI(3,4,5)P3 or an inhibitor of regulator of G-protein signaling 4 (RGS4). There was no difference in the effects of CCh on the hyperpolarization-activated current (If) between wildtype and Akita mice. Our study demonstrates that Akita diabetic mice demonstrate impaired parasympathetic regulation of HR and SAN function due to reduced responses of the SAN to parasympathetic agonists. Our experiments demonstrate a key role for insulin-dependent phosphoinositide 3-kinase (PI3K) signaling in the parasympathetic dysfunction seen in the SAN in diabetes.

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.