Ryanodine Receptor Stabilization Therapy Suppresses Ca 2+ -Based Arrhythmias in a Novel Model of Metabolic HFpEF.

Heart Failure with preserved ejection fraction (HFpEF) is the most prevalent form of heart failure worldwide and its significant mortality is associated with a high rate of sudden cardiac death (SCD; 30% - 40%). Chronic metabolic stress is an important driver of HFpEF, and clinical data show metabolic stress as a significant risk factor for ventricular arrhythmias in HFpEF patients. The mechanisms of SCD and ventricular arrhythmia in HFpEF remain critically understudied and empirical treatment is ineffective. To address this important knowledge gap, we developed a novel preclinical model of metabolic-stress induced HFpEF using Western diet (High fructose and fat) and hypertension induced by nitric oxide synthase inhibition (with L-NAME) in wildtype C57BL6/J mice. After 5 months, mice display all clinical characteristics of HFpEF and present with stress-induced sustained ventricular tachycardia (VT). Mechanistically, we found a novel pattern of arrhythmogenic intracellular Ca 2+ handling that is distinct from the well-characterized changes pathognomonic for heart failure with reduced ejection fraction. In addition, we show that the transverse tubular system remains intact in HFpEF and that arrhythmogenic, intracellular Ca 2+ mobilization becomes hyper-sensitive to ß- adrenergic activation. Finally, in proof-of-concept experiments we show in vivo that the clinically used intracellular calcium stabilizer dantrolene, which acts on the Ca 2+ release channels of the sarcoplasmic reticulum (SR), the ryanodine receptors, acutely prevents stress-induced VT in HFpEF mice. Therapeutic control of SR Ca 2+ leak may present a novel mechanistic treatment approach in metabolic HFpEF.

Na + /K + ATPase-Ca v 1.2 nanodomain differentially regulates intracellular [Na + ], [Ca 2+ ] and local adrenergic signaling in cardiac myocytes.

Background: The intracellular Na + concentration ([Na + ] i ) is a crucial but understudied regulator of cardiac myocyte function. The Na + /K + ATPase (NKA) controls the steady-state [Na + ] i and thereby determines the set-point for intracellular Ca 2+ . Here, we investigate the nanoscopic organization and local adrenergic regulation of the NKA macromolecular complex and how it differentially regulates the intracellular Na + and Ca 2+ homeostases in atrial and ventricular myocytes. Methods: Multicolor STORM super-resolution microscopy, Western Blot analyses, and in vivo examination of adrenergic regulation are employed to examine the organization and function of Na + nanodomains in cardiac myocytes. Quantitative fluorescence microscopy at high spatiotemporal resolution is used in conjunction with cellular electrophysiology to investigate intracellular Na + homeostasis in atrial and ventricular myocytes. Results: The NKAα1 (NKAα1) and the L-type Ca 2+ -channel (Ca v 1.2) form a nanodomain with a center-to center distance of ∼65 nm in both ventricular and atrial myocytes. NKAα1 protein expression levels are ∼3 fold higher in atria compared to ventricle. 100% higher atrial I NKA , produced by large NKA "superclusters", underlies the substantially lower Na + concentration in atrial myocytes compared to the benchmark values set in ventricular myocytes. The NKA's regulatory protein phospholemman (PLM) has similar expression levels across atria and ventricle resulting in a much lower PLM/NKAα1 ratio for atrial compared to ventricular tissue. In addition, a huge PLM phosphorylation reserve in atrial tissue produces a high ß-adrenergic sensitivity of I NKA in atrial myocytes. ß-adrenergic regulation of I NKA is locally mediated in the NKAα1-Ca v 1.2 nanodomain via A-kinase anchoring proteins. Conclusions: NKAα1, Ca v 1.2 and their accessory proteins form a structural and regulatory nanodomain at the cardiac dyad. The tissue-specific composition and local adrenergic regulation of this "signaling cloud" is a main regulator of the distinct global intracellular Na + and Ca 2+ concentrations in atrial and ventricular myocytes.

Calcium and bicarbonate signaling pathways have pivotal, resonating roles in matching ATP production to demand.

Mitochondrial ATP production in ventricular cardiomyocytes must be continually adjusted to rapidly replenish the ATP consumed by the working heart. Two systems are known to be critical in this regulation: mitochondrial matrix Ca2+ ([Ca2+]m) and blood flow that is tuned by local cardiomyocyte metabolic signaling. However, these two regulatory systems do not fully account for the physiological range of ATP consumption observed. We report here on the identity, location, and signaling cascade of a third regulatory system -- CO2/bicarbonate. CO2 is generated in the mitochondrial matrix as a metabolic waste product of the oxidation of nutrients. It is a lipid soluble gas that rapidly permeates the inner mitochondrial membrane and produces bicarbonate in a reaction accelerated by carbonic anhydrase. The bicarbonate level is tracked physiologically by a bicarbonate-activated soluble adenylyl cyclase (sAC). Using structural Airyscan super-resolution imaging and functional measurements we find that sAC is primarily inside the mitochondria of ventricular cardiomyocytes where it generates cAMP when activated by bicarbonate. Our data strongly suggest that ATP production in these mitochondria is regulated by this cAMP signaling cascade operating within the inter-membrane space by activating local EPAC1 (Exchange Protein directly Activated by cAMP) which turns on Rap1 (Ras-related protein-1). Thus, mitochondrial ATP production is increased by bicarbonate-triggered sAC-signaling through Rap1. Additional evidence is presented indicating that the cAMP signaling itself does not occur directly in the matrix. We also show that this third signaling process involving bicarbonate and sAC activates the mitochondrial ATP production machinery by working independently of, yet in conjunction with, [Ca2+]m-dependent ATP production to meet the energy needs of cellular activity in both health and disease. We propose that the bicarbonate and calcium signaling arms function in a resonant or complementary manner to match mitochondrial ATP production to the full range of energy consumption in ventricular cardiomyocytes.

Camera-based Measurements of Intracellular [Na+] in Murine Atrial Myocytes.

Intracellular sodium concentration ([Na+]i) is an important regulator of intracellular Ca2+. Its study provides insight into the activation of the sarcolemmal Na+/Ca2+ exchanger, the behavior of voltage-gated Na+ channels and the Na+,K+-ATPase. Intracellular Ca2+ signaling is altered in atrial diseases such as atrial fibrillation. While many of the mechanisms underlying altered intracellular Ca2+ homeostasis are characterized, the role of [Na+]i and its dysregulation in atrial pathologies is poorly understood. [Na+]i in atrial myocytes increases in response to increasing stimulation rates. Responsiveness to external field stimulation is therefore crucial for [Na+]i measurements in these cells. In addition, the long preparation (dye-loading) and experiment duration (calibration) require an isolation protocol that yields atrial myocytes of exceptional quality. Due to the small size of mouse atria and the composition of the intercellular matrix, the isolation of high quality adult murine atrial myocytes is difficult. Here, we describe an optimized Langendorff-perfusion based isolation protocol that consistently delivers a high yield of high quality atrial murine myocytes. Sodium-binding benzofuran isophthalate (SBFI) is the most commonly used fluorescent Na+ indicator. SBFI can be loaded into the cardiac myocyte either in its salt form through a glass pipette or as an acetoxymethyl (AM) ester that can penetrate the myocyte's sarcolemmal membrane. Intracellularly, SBFI-AM is de-esterified by cytosolic esterases. Due to variabilities in membrane penetration and cytosolic de-esterification each cell has to be calibrated in situ. Typically, measurements of [Na+]i using SBFI whole-cell epifluorescence are performed using a photomultiplier tube (PMT). This experimental set-up allows for only one cell to be measured at one time. Due to the length of myocyte dye loading and the calibration following each experiment data yield is low. We therefore developed an EMCCD camera-based technique to measure [Na+]i. This approach permits simultaneous [Na+]i measurements in multiple myocytes thus significantly increasing experimental yield.

Calcium Signaling Silencing in Atrial Fibrillation: Implications for Atrial Sodium Homeostasis.

Atrial fibrillation (AF) is the most common type of cardiac arrhythmia, affecting more than 33 million people worldwide. Despite important advances in therapy, AF's incidence remains high, and treatment often results in recurrence of the arrhythmia. A better understanding of the cellular and molecular changes that (1) trigger AF and (2) occur after the onset of AF will help to identify novel therapeutic targets. Over the past 20 years, a large body of research has shown that intracellular Ca2+ handling is dramatically altered in AF. While some of these changes are arrhythmogenic, other changes counteract cellular arrhythmogenic mechanisms (Calcium Signaling Silencing). The intracellular Na+ concentration ([Na+])i is a key regulator of intracellular Ca2+ handling in cardiac myocytes. Despite its importance in the regulation of intracellular Ca2+ handling, little is known about [Na+]i, its regulation, and how it might be changed in AF. Previous work suggests that there might be increases in the late component of the atrial Na+ current (INa,L) in AF, suggesting that [Na+]i levels might be high in AF. Indeed, a pharmacological blockade of INa,L has been suggested as a treatment for AF. Here, we review calcium signaling silencing and changes in intracellular Na+ homeostasis during AF. We summarize the proposed arrhythmogenic mechanisms associated with increases in INa,L during AF and discuss the evidence from clinical trials that have tested the pharmacological INa,L blocker ranolazine in the treatment of AF.

Attenuating persistent sodium current-induced atrial myopathy and fibrillation by preventing mitochondrial oxidative stress.

Mechanistically driven therapies for atrial fibrillation (AF), the most common cardiac arrhythmia, are urgently needed, the development of which requires improved understanding of the cellular signaling pathways that facilitate the structural and electrophysiological remodeling that occurs in the atria. Similar to humans, increased persistent Na+ current leads to the development of an atrial myopathy and spontaneous and long-lasting episodes of AF in mice. How increased persistent Na+ current causes both structural and electrophysiological remodeling in the atria is unknown. We crossbred mice expressing human F1759A-NaV1.5 channels with mice expressing human mitochondrial catalase (mCAT). Increased expression of mCAT attenuated mitochondrial and cellular reactive oxygen species (ROS) and the structural remodeling that was induced by persistent F1759A-Na+ current. Despite the heterogeneously prolonged atrial action potential, which was unaffected by the reduction in ROS, the incidences of spontaneous AF, pacing-induced after-depolarizations, and AF were substantially reduced. Expression of mCAT markedly reduced persistent Na+ current-induced ryanodine receptor oxidation and dysfunction. In summary, increased persistent Na+ current in atrial cardiomyocytes, which is observed in patients with AF, induced atrial enlargement, fibrosis, mitochondrial dysmorphology, early after-depolarizations, and AF, all of which can be attenuated by resolving mitochondrial oxidative stress.

Calcium influx through the mitochondrial calcium uniporter holocomplex, MCUcx.

Ca2+ flux into the mitochondrial matrix through the MCU holocomplex (MCUcx) has recently been measured quantitatively and with milliseconds resolution for the first time under physiological conditions in both heart and skeletal muscle. Additionally, the dynamic levels of Ca2+ in the mitochondrial matrix ([Ca2+]m) of cardiomyocytes were measured as it was controlled by the balance between influx of Ca2+ into the mitochondrial matrix through MCUcx and efflux through the mitochondrial Na+ / Ca2+ exchanger (NCLX). Under these conditions [Ca2+]m was shown to regulate ATP production by the mitochondria at only a few critical sites. Additional functions attributed to [Ca2+]m continue to be reported in the literature. Here we review the new findings attributed to MCUcx function and provide a framework for understanding and investigating mitochondrial Ca2+ influx features, many of which remain controversial. The properties and functions of the MCUcx subunits that constitute the holocomplex are challenging to tease apart. Such distinct subunits include EMRE, MCUR1, MICUx (i.e. MICU1, MICU2, MICU3), and the pore-forming subunits (MCUpore). Currently, the specific set of functions of each subunit remains non-quantitative and controversial. The more contentious issues are discussed in the context of the newly measured native MCUcx Ca2+ flux from heart and skeletal muscle. These MCUcx Ca2+ flux measurements have been shown to be a highly-regulated, tissue-specific with femto-Siemens Ca2+ conductances and with distinct extramitochondrial Ca2+ ([Ca2+]i) dependencies. These data from cardiac and skeletal muscle mitochondria have been examined quantitatively for their threshold [Ca2+]i levels and for hypothesized gatekeeping function and are discussed in the context of model cell (e.g. HeLa, MEF, HEK293, COS7 cells) measurements. Our new findings on MCUcx dependent matrix [Ca2+]m signaling provide a quantitative basis for on-going and new investigations of the roles of MCUcx in cardiac function ranging from metabolic fuel selection, capillary blood-flow control and the pathological activation of the mitochondrial permeability transition pore (mPTP). Additionally, this review presents the use of advanced new methods that can be readily adapted by any investigator to enable them to carry out quantitative Ca2+ measurements in mitochondria while controlling the inner mitochondrial membrane potential, ΔΨm.

The surprising complexity of KATP channel biology and of genetic diseases.

The ATP-sensitive K+ channel (KATP) is formed by the association of four inwardly rectifying K+ channel (Kir6.x) pore subunits with four sulphonylurea receptor (SUR) regulatory subunits. Kir6.x or SUR mutations result in KATP channelopathies, which reflect the physiological roles of these channels, including but not limited to insulin secretion, cardiac protection, and blood flow regulation. In this issue of the JCI, McClenaghan et al. explored one of the channelopathies, namely Cantu syndrome (CS), which is a result of one kind of KATP channel mutation. Using a knockin mouse model, the authors demonstrated that gain-of-function KATP mutations in vascular smooth muscle resulted in cardiac remodeling. Moreover, they were able to reverse the cardiovascular phenotypes by administering the KATP channel blocker glibenclamide. These results exemplify how genetic mutations can have an impact on developmental trajectories, and provide a therapeutic approach to mitigate cardiac hypertrophy in cases of CS.

Calcium signalling silencing in atrial fibrillation.

Subcellular calcium signalling silencing is a novel and distinct cellular and molecular adaptive response to rapid cardiac activation. Calcium signalling silencing develops during short-term sustained rapid atrial activation as seen clinically during paroxysmal atrial fibrillation (AF). It is the first 'anti-arrhythmic' adaptive response in the setting of AF and appears to counteract the maladaptive changes that lead to intracellular Ca2+ signalling instability and Ca2+ -based arrhythmogenicity. Calcium signalling silencing results in a failed propagation of the [Ca2+ ]i signal to the myocyte centre both in patients with AF and in a rabbit model. This adaptive mechanism leads to a substantial reduction in the expression levels of calcium release channels (ryanodine receptors, RyR2) in the sarcoplasmic reticulum, and the frequency of Ca2+ sparks and arrhythmogenic Ca2+ waves remains low. Less Ca2+ release per [Ca2+ ]i transient, increased fast Ca2+ buffering strength, shortened action potentials and reduced L-type Ca2+ current contribute to a substantial reduction of intracellular [Na+ ]. These features of Ca2+ signalling silencing are distinct and in contrast to the changes attributed to Ca2+ -based arrhythmogenicity. Some features of Ca2+ signalling silencing prevail in human AF suggesting that the Ca2+ signalling 'phenotype' in AF is a sum of Ca2+ stabilizing (Ca2+ signalling silencing) and Ca2+ destabilizing (arrhythmogenic unstable Ca2+ signalling) factors. Calcium signalling silencing is a part of the mechanisms that contribute to the natural progression of AF and may limit the role of Ca2+ -based arrhythmogenicity after the onset of AF.

Tachycardia-induced silencing of subcellular Ca2+ signaling in atrial myocytes.

Atrial fibrillation (AF) is characterized by sustained high atrial activation rates and arrhythmogenic cellular Ca2+ signaling instability; however, it is not clear how a high atrial rate and Ca2+ instability may be related. Here, we characterized subcellular Ca2+ signaling after 5 days of high atrial rates in a rabbit model. While some changes were similar to those in persistent AF, we identified a distinct pattern of stabilized subcellular Ca2+ signaling. Ca2+ sparks, arrhythmogenic Ca2+ waves, sarcoplasmic reticulum (SR) Ca2+ leak, and SR Ca2+ content were largely unaltered. Based on computational analysis, these findings were consistent with a higher Ca2+ leak due to PKA-dependent phosphorylation of SR Ca2+ channels (RyR2s), fewer RyR2s, and smaller RyR2 clusters in the SR. We determined that less Ca2+ release per [Ca2+]i transient, increased Ca2+ buffering strength, shortened action potentials, and reduced L-type Ca2+ current contribute to a stunning reduction of intracellular Na+ concentration following rapid atrial pacing. In both patients with AF and in our rabbit model, this silencing led to failed propagation of the [Ca2+]i signal to the myocyte center. We conclude that sustained high atrial rates alone silence Ca2+ signaling and do not produce Ca2+ signaling instability, consistent with an adaptive molecular and cellular response to atrial tachycardia.

Dynamic remodeling of intracellular Ca²âº signaling during atrial fibrillation.

During atrial fibrillation (AF) intracellular Ca(2+) signaling in atrial myocytes changes substantially. This 'remodeled' intracellular Ca(2+) homeostasis plays an important role in the development of the contractile dysfunction and the changes in atrial electrophysiology (contractile and electrical remodeling) that are characteristic of AF. Recent studies also show that unstable intracellular Ca(2+) signaling (i.e. increased Ca(2+) sparks and Ca(2+) waves) is present in atrial myocytes from AF patients and that it might contribute to cellular arrhythmogenic mechanisms that help maintain the arrhythmia. It is currently not well understood how and when unstable Ca(2+) signaling develops during the progression of AF, or if, in cases of structural heart disease, it even precedes the onset of AF. Current work therefore in particular aims to elucidate the molecular and sub-cellular mechanisms underlying the arrhythmogenic intracellular Ca(2+) signaling instability in AF. As treatment of AF remains difficult, the identification of novel targets for counteracting or preventing arrhythmogenic Ca(2+) signaling is an important part of AF research. It is therefore important to recognize which phase of AF is addressed in a specific research (and ultimately treatment) approach. Here we review and critique the distinct alterations in intracellular Ca(2+) signaling during the progression of AF from initial intracellular Ca(2+) overload to the remodeling process. We address Ca(2+) signaling after cardioversion of the arrhythmia and its potential role in the recurrence of AF. We propose that altered Ca(2+) signaling during AF progression consists of three phases 1.) Ca(2+) Overload, 2.) Remodeling, and 3.) Steady State. Similarly, after AF termination three distinct phases of 'recovery' of intracellular Ca(2+) handling occur. 4.) Calcium Unloading, 5.) Reverse Remodeling and 6.) Full Recovery. While there is evidence that unstable Ca(2+) signaling is part of phases 1, 3 and 4, phase 2 (remodeling) appears to have a more stabilizing function on Ca(2+) signaling ('Ca(2+) silencing'). This has important implications for the timing and type of pharmacological intervention, especially for new compounds aimed at intracellular 'Ca(2+) stabilization'.

Alterations of atrial Ca(2+) handling as cause and consequence of atrial fibrillation.

Atrial fibrillation (AF) is the most prevalent sustained arrhythmia. As the most important risk factor for embolic stroke, AF is associated with a high morbidity and mortality. Despite decades of research, successful (pharmacological and interventional) 'ablation' of the arrhythmia remains challenging. AF is characterized by a diverse aetiology, including heart failure, hypertension, and valvular disease. Based on this understanding, new treatment strategies that are specifically tailored to the underlying pathophysiology of a certain 'type' of AF are being developed. One important aspect of AF pathophysiology is altered intracellular Ca(2+) handling. Due to the increase in the atrial activation rate and the subsequent initial [Ca(2+)](i) overload, AF induces 'remodelling' of intracellular Ca(2+) handling. Current research focuses on unravelling the contribution of altered intracellular Ca(2+) handling to different types of AF. More specifically, changes in intracellular Ca(2+) homeostasis preceding the onset of AF, in conditions which predispose to AF (e.g. heart failure), appear to be different from changes in Ca(2+) handling developing after the onset of AF. Here we review and critique altered intracellular Ca(2+) handling and its contribution to three specific aspects of AF pathophysiology, (i) excitation-transcription coupling and Ca(2+)-dependent signalling pathways, (ii) atrial contractile dysfunction, and (iii) arrhythmogenicity.

Multiple potential molecular contributors to atrial hypocontractility caused by atrial tachycardia remodeling in dogs.

BACKGROUND: Atrial fibrillation impairs atrial contractility, inducing atrial stunning that promotes thromboembolic stroke. Action potential (AP)-prolonging drugs are reported to normalize atrial hypocontractility caused by atrial tachycardia remodeling (ATR). Here, we addressed the role of AP duration (APD) changes in ATR-induced hypocontractility. METHODS AND RESULTS: ATR (7-day tachypacing) decreased APD (perforated patch recording) by ≈50%, atrial contractility (echocardiography, cardiomyocyte video edge detection), and [Ca(2+)](i) transients. ATR AP waveforms suppressed [Ca(2+)](i) transients and cell shortening of control cardiomyocytes; whereas control AP waveforms improved [Ca(2+)](i) transients and cell shortening in ATR cells. However, ATR cardiomyocytes clamped with the same control AP waveform had ≈60% smaller [Ca(2+)](i) transients and cell shortening than control cells. We therefore sought additional mechanisms of contractile impairment. Whole-cell voltage clamp revealed reduced I(CaL); I(CaL) inhibition superimposed on ATR APs further suppressed [Ca(2+)](i) transients in control cells. Confocal microscopy indicated ATR-impaired propagation of the Ca(2+) release signal to the cell center in association with loss of t-tubular structures. Myofilament function studies in skinned permeabilized cardiomyocytes showed altered Ca(2+) sensitivity and force redevelopment in ATR, possibly due to hypophosphorylation of myosin-binding protein C and myosin light-chain protein 2a (immunoblot). Hypophosphorylation was related to multiple phosphorylation system abnormalities where protein kinase A regulatory subunits were downregulated, whereas autophosphorylation and expression of Ca(2+)-calmodulin-dependent protein kinase IIδ and protein phosphatase 1 activity were enhanced. Recovery of [Ca(2+)](i) transients and cell shortening occurred in parallel after ATR cessation. CONCLUSIONS: Shortening of APD contributes to hypocontractility induced by 1-week ATR but accounts for it only partially. Additional contractility-suppressing mechanisms include I(CaL) current reduction, impaired subcellular Ca(2+) signal transmission, and altered myofilament function associated with abnormal myosin and myosin-associated protein phosphorylation. The complex mechanistic basis of the atrial hypocontractility associated with AF argues for upstream therapeutic targeting rather than interventions directed toward specific downstream pathophysiological derangements.

Distinct contractile and molecular differences between two goat models of atrial dysfunction: AV block-induced atrial dilatation and atrial fibrillation.

Atrial dilatation is an independent risk factor for thromboembolism in patients with and without atrial fibrillation (AF). In many patients, atrial dilatation goes along with depressed contractile function of the dilated atria. While some mechanisms causing atrial contractile dysfunction in fibrillating atria have been addressed previously, the cellular and molecular mechanisms of atrial contractile remodeling in dilated atria are unknown. This study characterized in vivo atrial contractile function in a goat model of atrial dilatation and compared it to a goat model of AF. Differences in the underlying mechanisms were elucidated by studying contractile function, electrophysiology and sarcoplasmic reticulum (SR) Ca2+ load in atrial muscle bundles and by analyzing expression and phosphorylation levels of key Ca2+-handling proteins, myofilaments and the expression and activity of their upstream regulators. In 7 chronically instrumented, awake goats atrial contractile dysfunction was monitored during 3 weeks of progressive atrial dilatation after AV-node ablation (AV block goats (AVB)). In open chest experiments atrial work index (AWI) and refractoriness were measured (10 goats with AVB, 5 goats with ten days of AF induced by repetitive atrial burst pacing (AF), 10 controls). Isometric force of contraction (FC), transmembrane action potentials (APs) and rapid cooling contractures (RCC, a measure of SR Ca2+ load) were studied in right atrial muscle bundles. Total and phosphorylated Ca2+-handling and myofilament protein levels were quantified by Western blot. In AVB goats, atrial size increased by 18% (from 26.6+/-4.4 to 31.6+/-5.5 mm, n=7 p<0.01) while atrial fractional shortening (AFS) decreased (from 18.4+/-1.7 to 12.8+/-4.0% at 400 ms, n=7, p<0.01). In open chest experiments, AWI was reduced in AVB and in AF goats compared to controls (at 400 ms: 8.4+/-0.9, n=7, and 3.2+/-1.8, n=5, vs 18.9+/-5.3 mmxmmHg, n=7, respectively, p<0.05 vs control). FC of isolated right atrial muscle bundles was reduced in AVB (n=8) and in AF (n=5) goats compared to controls (n=9) (at 2 Hz: 2.3+/-0.5 and 0.7+/-0.2 vs 5.5+/-1.0 mN/mm2, respectively, p<0.05). APs were shorter in AF, but unchanged in AVB goats. RCCs were reduced in AVB and AF versus control (AVB, 3.4+/-0.5 and AF, 4.1+/-1.4 vs 12.2+/-3.2 mN/mm2, p<0.05). Protein levels of protein kinase A (PKA) phosphorylated phospholamban (PLB) were reduced in AVB (n=8) and AF (n=8) vs control (n=7) by 37.9+/-12.4% and 29.7+/-10.1%, respectively (p<0.01), whereas calmodulin-dependent protein kinase II (CaMKII) phosphorylated ryanodine channels (RyR2) were increased by 166+/-55% in AVB (n=8) and by 146+/-56% in AF (n=8) goats (p<0.01). PKA-phosphorylated myosin-binding protein-C and troponin-I were reduced exclusively in AVB goat atria (by 75+/-10% and 55+/-15%, respectively, n=8, p<0.05). Atrial dilatation developing during slow ventricular rhythm after complete AV block as well as AF-induced remodeling are associated with atrial contractile dysfunction. Both AVB and AF goat atria show decreased SR Ca2+ load, likely caused by PLB dephosphorylation and RYR2 hyperphosphorylation. While shorter APs further compromise contractility in AF goat atria, reduced myofilament phosphorylation may impair contractility in AVB goat atria. Thus, atrial hypocontractility appears to have distinct molecular contributors in different types of atrial remodeling.

Pharmacological evidence for altered src kinase regulation of I (Ca,L) in patients with chronic atrial fibrillation.

A reduction in L-type Ca(2+) current (I (Ca,L)) contributes to electrical remodeling in chronic atrial fibrillation (AF). Whether the decrease in I (Ca,L) is solely due to a reduction in channel proteins remains controversial. Protein tyrosine kinases (PTK) have been described as potent modulators of I (Ca,L) in cardiomyocytes. We studied alpha(1C) L-type Ca(2+) channel subunit expression and the regulation of I (Ca,L) by PTK in chronic AF using PTK inhibitors: genistein, a nonselective inhibitor of PTK, and 4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo-3,4-d-pyrimidine (PP1), a selective inhibitor of src kinases. Furthermore, type-1 and type-2A protein phosphatase activity was measured with phosphorylase as substrate in whole-cell lysates derived from atrial tissue of AF patients. Right atrial appendages were obtained from patients undergoing open-heart surgery. Protein levels of alpha(1C) L-type Ca(2+) channel subunit were determined using Western blot analysis and normalized to the protein amounts of calsequestrin as internal control. The protein concentrations of alpha(1C) did not differ between AF and sinus rhythm (SR; alpha(1C)/calsequestrin: 1.0 +/- 0.1 and 1.2 +/- 0.2, respectively, n = 8 patients). In cardiomyocytes from patients in SR (n = 20 patients), genistein and PP1 both evoked similar increases in I (Ca,L) from 3.0 +/- 0.3 to 6.1 +/- 0.8 pA/pF and from 2.8 +/- 0.4 to 6.1 +/- 0.6 pA/pF, respectively. In cells from AF patients (n = 10 patients), basal I (Ca,L) was significantly lower. In this case, genistein lead to the same relative increase in I (Ca,L) as in SR cells (from 1.46 +/- 0.30 to 3.2 +/- 1.0 pA/pF), whereas no increase was elicited by PP1 suggesting impaired regulation of I (Ca,L) by src kinases in AF. Total and type 1 and type 2A-related phosphatase activities were higher in tissue from patients with chronic AF compared to SR (4.8 +/- 0.4, 2.1 +/- 0.2, and 2.7 +/- 0.4 nmol/mg/min and 3.6 +/- 0.4, 1.3 +/- 0.2, and 2.4 +/- 0.3 nmol/mg/min, respectively, n = 7 patients per group). Downregulation of I (Ca,L) in AF is not due to a reduction in L-type Ca(2+) channel protein expression. Indirect evidence for an impaired src kinase regulation of I (Ca,L) together with an increased phosphatase activity suggests that a complex alteration in the kinase/phosphatase balance leads to I (Ca,L) dysregulation in chronic AF.

Blockade of atrial-specific K+-currents increases atrial but not ventricular contractility by enhancing reverse mode Na+/Ca2+-exchange.

BACKGROUND: AVE0118 (2'-{[2-(4-Methoxy-phenyl)-acetylamino]-methyl}-biphenyl-2-carboxylic acid (2-pyridin-3-yl-ethyl)-amide) blocks atrial ultrarapid delayed rectifier currents (I(Kur)) and prolongs the atrial action potential (AP) plateau without affecting ventricular repolarisation. In patients with atrial contractile dysfunction due to atrial tachyarrhythmias, this response might increase atrial contractility without risk of ventricular proarrhythmia. This study was designed to evaluate the inotropic mechanisms of AVE0118. METHODS AND RESULTS: In isometrically contracting atrial trabeculae, AVE0118 increased contractile force by 55.4% in sinus rhythm patients (n = 9) and by 107.4% in patients with atrial fibrillation (n = 8). In freshly isolated canine atrial myocytes studied under perforated patch current clamp (37 degrees C), AVE0118 increased myocyte fractional shortening from 3.8+/-0.6 to 9.6+/-0.8% and prolonged action potential duration at 30% repolarisation from 9+/-2 to 102+/-11 ms. Clamping cells to an AP waveform recorded during exposure to AVE0118 produced the same inotropic response as the drug itself. In action potential clamp, peak Ca2+ inward current (I(CaL)) current declined from 5.5+/-1.3 pA/pF during control to 4.1+/-0.7 pA/pF when an AP recorded in the presence of AVE0118 was used as command waveform. However, I(CaL) was more sustained with AVE0118 and the time integral did not change (135+/-37 vs. 173+/-30 pA/pFms, p = ns). Importantly, blockade of reverse mode Na+/Ca2+-exchanger activity with 5 microM KBR7943 or using a Na+-free pipette solution abolished the positive inotropic effect of the AP recorded in the presence of AVE0118. In ventricular myocytes AVE0118 did not elicit a positive inotropic response. CONCLUSIONS: Block of I(Kur) by AVE0118 enhances atrial contractility both in patients with sinus rhythm and atrial fibrillation. The positive inotropic effect is atrial-specific and due to the changes of the action potential configuration which enhances Ca2+ entry via reverse mode Na+/Ca2+ exchange.

AVE0118, blocker of the transient outward current (I(to)) and ultrarapid delayed rectifier current (I(Kur)), fully restores atrial contractility after cardioversion of atrial fibrillation in the goat.

BACKGROUND: The loss of atrial contractile function after cardioversion of atrial fibrillation (AF) contributes to the thromboembolic risk associated with AF. The newly developed blocker of the transient outward current (I(to)) and ultrarapid delayed rectifier current (I(Kur)) AVE0118 prolongs atrial action potential duration and might therefore enhance atrial contractility. We compared the ability of AVE0118 to restore atrial contraction after cardioversion of AF with the efficacy of conventional positive inotropic compounds in the goat model of AF. METHODS AND RESULTS: Eighteen goats were chronically instrumented with epicardial electrodes, a pressure transducer in the right atrium, and piezoelectric crystals to measure right atrial diameter. Atrial contractility and refractoriness and QT duration were measured before and after 1 week (3 to 8 days) of AF induced by repetitive burst pacing. The measurements were repeated after administration of digoxin (0.02 mg/kg), dobutamine (5 microg x kg(-1) x min(-1)), the Ca2+ sensitizer EMD57033 (1 mg x kg(-1) x min(-1)), the L-type Ca2+ channel agonist BayY5959 (0.1 mg x kg(-1) x min(-1)), and AVE0118 (0.01 to 0.2 mg x kg(-1) x min(-1)). The effect of AVE0118 on the configuration of atrial monophasic action potentials was determined for comparison. After 1 week of AF, atrial contractility during sinus rhythm or slow atrial pacing was reduced to <10%. Digoxin and dobutamine failed to increase atrial contractility. EMD57033 restored 41% and BayY5959 restored 48% of atrial contractility at baseline. BayY5959 significantly prolonged QT duration by 24.7%. AVE0118 enhanced atrial contraction to 156% of the baseline value. The positive inotropic effect was accompanied by a pronounced prolongation of atrial action potential duration and refractoriness, whereas QT duration remained unchanged. CONCLUSIONS: Conventional positive inotropic drugs showed limited effect on atrial contractility after cardioversion of AF or produced QT prolongation. In contrast, the I(to)/I(Kur) blocker AVE0118 fully restored atrial contraction without proarrhythmic effects on the ventricle.

The L-type Ca2+-channel subunits alpha1C and beta2 are not downregulated in atrial myocardium of patients with chronic atrial fibrillation.

OBJECTIVE: Electrical remodeling as well as atrial contractile dysfunction after the conversion of atrial fibrillation (AF) to sinus rhythm (SR) are mainly caused by a reduction of the inward L-type Ca(2+) current (I(CaL)). We investigated whether the expression of L-type Ca2+-channel subunits was reduced in atrial myocardium of AF patients. METHODS: Right atrial appendages were obtained from patients undergoing coronary artery bypass graft surgery (CAD, n = 35) or mitral valve surgery (MVD, n = 37). Seventeen of the CAD patients and 18 of the MVD patients were in chronic (>3 months) AF, whereas the others were in SR. The protein expression of the L-type Ca2+-channel subunits alpha1C and beta2 was quantified by western blot analysis. Furthermore, we measured the density of dihydropyridine (DHP)-binding sites of the L-type Ca2+ channel using 3H-PN220-100 as radioligand. RESULTS: Surprisingly, the alpha1C and the beta2-subunit expression was not altered in atrial myocardium of AF patients. Also, the DHP-binding site density was unchanged. CONCLUSION: The protein expression of the L-type Ca2+-channel subunits alpha1C or beta2 is not reduced in atrial myocardium of AF patients. Therefore, the reduced I(CaL) might be due to downregulation of other accessory subunits (alpha2delta), expression of aberrant subunits, changes in channel trafficking or alterations in channel function.

Atrial fibrillation-induced atrial contractile dysfunction: a tachycardiomyopathy of a different sort.

OBJECTIVE: Although AF-induced atrial contractile dysfunction has significant clinical implications the underlying intracellular mechanisms are poorly understood. METHODS: From the right atrial appendages of 59 consecutive patients undergoing mitral valve surgery (31 in SR, 28 in chronic AF) thin muscle preparations (diameter<0.7 mm) were isolated. Isometric force of contraction was measured in the presence of different concentrations of Ca(2+) and isoprenaline. To assess the function of the sarcoplasmic reticulum, the force-frequency relationship and the post-rest potentiation were studied. The myocardial density of the ryanodine-sensitive calcium release channel (CRC) of the sarcoplasmic reticulum was determined by [3H]ryanodine binding. Myocardial content of SR-Ca(2+)-ATPase (SERCA), phospholamban (Plb), calsequestrin (Cals) and the Na(+)/Ca(2+)-exchanger (NCX) were analyzed by Western blot analysis. Adenylyl cyclase activity was measured with a radiolabeled bioassay using [32P]ATP as a tracer. RESULTS: In 72 muscle preparations of SR patients contractile force was 10.9+/-1.8 mN/mm(2) compared to 3.3+/-0.9 mN/mm(2) (n=48, P<0.01) in AF patients. The positive inotropic effect of isoprenaline was diminished but the stimulatory effect on relaxation and the adenylyl cyclase were not altered in AF patients. The force-frequency relation and the post-rest potentiation were enhanced in atrial myocardium of AF patients. The protein levels of CRC, SERCA, Plb, and Cals were not different between the two groups. In contrast, the Na(+)/Ca(2+)-exchanger was upregulated by 67% in atria of AF patients. CONCLUSIONS: AF-induced atrial contractile dysfunction is not due to beta-adrenergic desensitization or dysfunction of the sarcoplasmic reticulum and thus is based on different cellular mechanisms than a ventricular tachycardia-induced cardiomyopathy. Instead, downregulation or altered function of the L-type Ca(2+)-channel and an increased Ca(2+) extrusion via the Na(+)/Ca(2+)-exchanger seem to be responsible for the depressed contractility in remodeled atria.