Molecular and cellular neurocardiology in heart disease.

This paper updates and builds on a previous White Paper in this journal that some of us contributed to concerning the molecular and cellular basis of cardiac neurobiology of heart disease. Here we focus on recent findings that underpin cardiac autonomic development, novel intracellular pathways and neuroplasticity. Throughout we highlight unanswered questions and areas of controversy. Whilst some neurochemical pathways are already demonstrating prognostic viability in patients with heart failure, we also discuss the opportunity to better understand sympathetic impairment by using patient specific stem cells that provides pathophysiological contextualization to study 'disease in a dish'. Novel imaging techniques and spatial transcriptomics are also facilitating a road map for target discovery of molecular pathways that may form a therapeutic opportunity to treat cardiac dysautonomia.

The heart-bone connection: relationships between myocardial infarction and osteoporotic fracture.

Myocardial infarction (MI) and osteoporotic fracture (Fx) are two of the leading causes of mortality and morbidity worldwide. Although these traumatic injuries are treated as if they are independent, there is epidemiological evidence linking the incidence of Fx and MI, thus raising the question of whether each of these events can actively influence the risk of the other. Atherosclerotic cardiovascular disease and osteoporosis, the chronic conditions leading to MI and Fx, are known to have shared pathoetiology. Furthermore, sustained systemic inflammation after traumas such as MI and Fx has been shown to exacerbate both underlying chronic conditions. However, the effects of MI and Fx outside their own system have not been well studied. The sympathetic nervous system (SNS) and the complement system initiate a systemic response after MI that could lead to subsequent changes in bone remodeling through osteoclasts. Similarly, SNS and complement system activation following fracture could lead to heart tissue damage and exacerbate atherosclerosis. To determine whether damaging bone-heart cross talk may be important comorbidity following Fx or MI, this review details the current understanding of bone loss after MI, cardiovascular damage after Fx, and possible shared underlying mechanisms of these processes.

Sex as a biological variable for cardiovascular physiology.

There have been ongoing efforts by federal agencies and scientific communities since the early 1990s to incorporate sex and/or gender in all aspects of cardiovascular research. Scientific journals provide a critical function as change agents to influence transformation by encouraging submissions for topic areas, and by setting standards and expectations for articles submitted to the journal. As part of ongoing efforts to advance sex and gender in cardiovascular physiology research, the American Journal of Physiology-Heart and Circulatory Physiology recently launched a call for papers on Considering Sex as a Biological Variable. This call was an overwhelming success, resulting in 78 articles published in this collection. This review summarizes the major themes of the collection, including Sex as a Biological Variable Within: Endothelial Cell and Vascular Physiology, Cardiovascular Immunity and Inflammation, Metabolism and Mitochondrial Energy, Extracellular Matrix Turnover and Fibrosis, Neurohormonal Signaling, and Cardiovascular Clinical and Epidemiology Assessments. Several articles also focused on establishing rigor and reproducibility of key physiological measurements involved in cardiovascular health and disease, as well as recommendations and considerations for study design. Combined, these articles summarize our current understanding of sex and gender influences on cardiovascular physiology and pathophysiology and provide insight into future directions needed to further expand our knowledge.

Sympathetic structural and electrophysiological remodeling in a rabbit model of reperfused myocardial infarction.

Chondroitin sulfate proteoglycans (CSPGs) inhibit sympathetic reinnervation in rodent hearts post myocardial infarction (MI), causing regional hypo-innervation that is associated with supersensitivity of ß-adrenergic receptors and increased arrhythmia susceptibility. To investigate the role of CSPGs and hypo-innervation in the heart of larger mammals, we used a rabbit model of reperfused MI and tested electrophysiological responses to sympathetic nerve stimulation (SNS). Innervated hearts from MI and sham rabbits were optically mapped using voltage and Ca2+-sensitive dyes. SNS was performed with electrical stimulation of the spinal cord and ß-adrenergic responsiveness was tested using isoproterenol. Sympathetic nerve density and CSPG expression were evaluated using immunohistochemistry. CSPGs were robustly expressed in the infarct region of all MI hearts, and the presence of CSPGs was associated with reduced sympathetic nerve density in the infarct vs. remote region. Action potential duration (APD) dispersion and susceptibility to ventricular tachycardia/fibrillation (VT/VF) were increased with SNS in MI hearts but not in sham. SNS decreased APD80 in MI but not sham hearts, while isoproterenol decreased APD80 in both groups. Isoproterenol also shortened Ca2+ transient duration (CaTD80) in both groups but to a greater extent in MI hearts. Our data suggest sympathetic remodeling post-MI is similar between rodents and rabbits, with CSPGs associated with sympathetic hypo-innervation. Despite a reduction in sympathetic nerve density, the infarct region of MI hearts remained responsive to both physiological SNS and isoproterenol, potentially through preserved or elevated ß-adrenergic responsiveness, which may underly increased APD dispersion and susceptibility for VT/VF.

Chronic nicotine exposure is associated with electrophysiological and sympathetic remodeling in the intact rabbit heart.

Nicotine is the primary addictive component of tobacco products. Through its actions on the heart and autonomic nervous system, nicotine exposure is associated with electrophysiological changes and increased arrhythmia susceptibility. To assess the underlying mechanisms, we treated rabbits with transdermal nicotine (NIC, 21 mg/day) or control (CT) patches for 28 days before performing dual optical mapping of transmembrane potential (RH237) and intracellular Ca2+ (Rhod-2 AM) in isolated hearts with intact sympathetic innervation. Sympathetic nerve stimulation (SNS) was performed at the first to third thoracic vertebrae, and ß-adrenergic responsiveness was additionally evaluated following norepinephrine (NE) perfusion. Baseline ex vivo heart rate (HR) and SNS stimulation threshold were higher in NIC versus CT (P = 0.004 and P = 0.003, respectively). Action potential duration alternans emerged at longer pacing cycle lengths (PCL) in NIC versus CT at baseline (P = 0.002) and during SNS (P = 0.0003), with similar results obtained for Ca2+ transient alternans. SNS shortened the PCL at which alternans emerged in CT but not in NIC hearts. NIC-exposed hearts tended to have slower and reduced HR responses to NE perfusion, but ventricular responses to NE were comparable between groups. Although fibrosis was unaltered, NIC hearts had lower sympathetic nerve density (P = 0.03) but no difference in NE content versus CT. These results suggest both sympathetic hypoinnervation of the myocardium and regional differences in ß-adrenergic responsiveness with NIC. This autonomic remodeling may contribute to the increased risk of arrhythmias associated with nicotine exposure, which may be further exacerbated with long-term use.NEW & NOTEWORTHY Here, we show that chronic nicotine exposure was associated with increased heart rate, increased susceptibility to alternans, and reduced sympathetic electrophysiological responses in the intact rabbit heart. We suggest that this was due to sympathetic hypoinnervation of the myocardium and diminished ß-adrenergic responsiveness of the sinoatrial node following nicotine treatment. Though these differences did not result in increased arrhythmia propensity in our study, we hypothesize that prolonged nicotine exposure may exacerbate this proarrhythmic remodeling.

A multiscale predictive digital twin for neurocardiac modulation.

Cardiac function is tightly regulated by the autonomic nervous system (ANS). Activation of the sympathetic nervous system increases cardiac output by increasing heart rate and stroke volume, while parasympathetic nerve stimulation instantly slows heart rate. Importantly, imbalance in autonomic control of the heart has been implicated in the development of arrhythmias and heart failure. Understanding of the mechanisms and effects of autonomic stimulation is a major challenge because synapses in different regions of the heart result in multiple changes to heart function. For example, nerve synapses on the sinoatrial node (SAN) impact pacemaking, while synapses on contractile cells alter contraction and arrhythmia vulnerability. Here, we present a multiscale neurocardiac modelling and simulator tool that predicts the effect of efferent stimulation of the sympathetic and parasympathetic branches of the ANS on the cardiac SAN and ventricular myocardium. The model includes a layered representation of the ANS and reproduces firing properties measured experimentally. Model parameters are derived from experiments and atomistic simulations. The model is a first prototype of a digital twin that is applied to make predictions across all system scales, from subcellular signalling to pacemaker frequency to tissue level responses. We predict conditions under which autonomic imbalance induces proarrhythmia and can be modified to prevent or inhibit arrhythmia. In summary, the multiscale model constitutes a predictive digital twin framework to test and guide high-throughput prediction of novel neuromodulatory therapy. KEY POINTS: A multi-layered model representation of the autonomic nervous system that includes sympathetic and parasympathetic branches, each with sparse random intralayer connectivity, synaptic dynamics and conductance based integrate-and-fire neurons generates firing patterns in close agreement with experiment. A key feature of the neurocardiac computational model is the connection between the autonomic nervous system and both pacemaker and contractile cells, where modification to pacemaker frequency drives initiation of electrical signals in the contractile cells. We utilized atomic-scale molecular dynamics simulations to predict the association and dissociation rates of noradrenaline with the ß-adrenergic receptor. Multiscale predictions demonstrate how autonomic imbalance may increase proclivity to arrhythmias or be used to terminate arrhythmias. The model serves as a first step towards a digital twin for predicting neuromodulation to prevent or reduce disease.

Diversity of cells and signals in the cardiovascular system.

This white paper is the outcome of the seventh UC Davis Cardiovascular Research Symposium on Systems Approach to Understanding Cardiovascular Disease and Arrhythmia. This biannual meeting aims to bring together leading experts in subfields of cardiovascular biomedicine to focus on topics of importance to the field. The theme of the 2022 Symposium was 'Cell Diversity in the Cardiovascular System, cell-autonomous and cell-cell signalling'. Experts in the field contributed their experimental and mathematical modelling perspectives and discussed emerging questions, controversies, and challenges in examining cell and signal diversity, co-ordination and interrelationships involved in cardiovascular function. This paper originates from the topics of formal presentations and informal discussions from the Symposium, which aimed to develop a holistic view of how the multiple cell types in the cardiovascular system integrate to influence cardiovascular function, disease progression and therapeutic strategies. The first section describes the major cell types (e.g. cardiomyocytes, vascular smooth muscle and endothelial cells, fibroblasts, neurons, immune cells, etc.) and the signals involved in cardiovascular function. The second section emphasizes the complexity at the subcellular, cellular and system levels in the context of cardiovascular development, ageing and disease. Finally, the third section surveys the technological innovations that allow the interrogation of this diversity and advancing our understanding of the integrated cardiovascular function and dysfunction.

CaMKII Serine 280 O-GlcNAcylation Links Diabetic Hyperglycemia to Proarrhythmia.

[Figure: see text].

Autonomic control of ventricular function in health and disease: current state of the art.

PURPOSE: Cardiac autonomic dysfunction is one of the main pillars of cardiovascular pathophysiology. The purpose of this review is to provide an overview of the current state of the art on the pathological remodeling that occurs within the autonomic nervous system with cardiac injury and available neuromodulatory therapies for autonomic dysfunction in heart failure. METHODS: Data from peer-reviewed publications on autonomic function in health and after cardiac injury are reviewed. The role of and evidence behind various neuromodulatory therapies both in preclinical investigation and in-use in clinical practice are summarized. RESULTS: A harmonic interplay between the heart and the autonomic nervous system exists at multiple levels of the neuraxis. This interplay becomes disrupted in the setting of cardiovascular disease, resulting in pathological changes at multiple levels, from subcellular cardiac signaling of neurotransmitters to extra-cardiac, extra-thoracic remodeling. The subsequent detrimental cycle of sympathovagal imbalance, characterized by sympathoexcitation and parasympathetic withdrawal, predisposes to ventricular arrhythmias, progression of heart failure, and cardiac mortality. Knowledge on the etiology and pathophysiology of this condition has increased exponentially over the past few decades, resulting in a number of different neuromodulatory approaches. However, significant knowledge gaps in both sympathetic and parasympathetic interactions and causal factors that mediate progressive sympathoexcitation and parasympathetic dysfunction remain. CONCLUSIONS: Although our understanding of autonomic imbalance in cardiovascular diseases has significantly increased, specific, pivotal mediators of this imbalance and the recognition and implementation of available autonomic parameters and neuromodulatory therapies are still lagging.

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.

Stop the beat to see the rhythm: excitation-contraction uncoupling in cardiac research.

Optical mapping is an imaging technique that is extensively used in cardiovascular research, wherein parameter-sensitive fluorescent indicators are used to study the electrophysiology and excitation-contraction coupling of cardiac tissues. Despite many benefits of optical mapping, eliminating motion artifacts within the optical signals is a major challenge, as myocardial contraction interferes with the faithful acquisition of action potentials and intracellular calcium transients. As such, excitation-contraction uncoupling agents are frequently used to reduce signal distortion by suppressing contraction. When compared with other uncoupling agents, blebbistatin is the most frequently used, as it offers increased potency with minimal direct effects on cardiac electrophysiology. Nevertheless, blebbistatin may exert secondary effects on electrical activity, metabolism, and coronary flow, and the incorrect administration of blebbistatin to cardiac tissue can prove detrimental, resulting in erroneous interpretation of optical mapping results. In this "Getting It Right" perspective, we briefly review the literature regarding the use of blebbistatin in cardiac optical mapping experiments, highlight potential secondary effects of blebbistatin on cardiac electrical activity and metabolic demand, and conclude with the consensus of the authors on best practices for effectively using blebbistatin in optical mapping studies of cardiac tissue.

Reperfused vs. nonreperfused myocardial infarction: when to use which model.

There is a lack of understanding in the cardiac remodeling field regarding the use of nonreperfused myocardial infarction (MI) and reperfused MI in animal models of MI. This Perspectives summarizes the consensus of the authors regarding how to select the optimum model for your experiments and is a part of ongoing efforts to establish rigor and reproducibility in cardiac physiology research.

Guidelines for in vivo mouse models of myocardial infarction.

Despite significant improvements in reperfusion strategies, acute coronary syndromes all too often culminate in a myocardial infarction (MI). The consequent MI can, in turn, lead to remodeling of the left ventricle (LV), the development of LV dysfunction, and ultimately progression to heart failure (HF). Accordingly, an improved understanding of the underlying mechanisms of MI remodeling and progression to HF is necessary. One common approach to examine MI pathology is with murine models that recapitulate components of the clinical context of acute coronary syndrome and subsequent MI. We evaluated the different approaches used to produce MI in mouse models and identified opportunities to consolidate methods, recognizing that reperfused and nonreperfused MI yield different responses. The overall goal in compiling this consensus statement is to unify best practices regarding mouse MI models to improve interpretation and allow comparative examination across studies and laboratories. These guidelines will help to establish rigor and reproducibility and provide increased potential for clinical translation.

Cardiac sympathetic nerve transdifferentiation reduces action potential heterogeneity after myocardial infarction.

Cardiac sympathetic nerves undergo cholinergic transdifferentiation following reperfused myocardial infarction (MI), whereby the sympathetic nerves release both norepinephrine (NE) and acetylcholine (ACh). The functional electrophysiological consequences of post-MI transdifferentiation have never been explored. We performed MI or sham surgery in wild-type (WT) mice and mice in which choline acetyltransferase was deleted from adult noradrenergic neurons [knockout (KO)]. Electrophysiological activity was assessed with optical mapping of action potentials (AP) and intracellular Ca2+ transients (CaT) in innervated Langendorff-perfused hearts. KO MI hearts had similar NE content but reduced ACh content compared with WT MI hearts (0.360 ± 0.074 vs. 0.493 ± 0.087 pmol/mg; KO, n = 6; WT, n = 4; P < 0.05). KO MI hearts also had higher basal ex vivo heart rates versus WT MI hearts (328.5 ± 35.3 vs. 247.4 ± 62.4 beats/min; KO, n = 8; WT, n = 6; P < 0.05). AP duration at 80% repolarization was significantly shorter in the remote and border zones of KO MI versus WT MI hearts, whereas AP durations (APDs) were similar in infarct regions. This APD heterogeneity resulted in increased APD dispersion in the KO MI versus WT MI hearts (11.9 ± 2.7 vs. 8.2 ± 2.3 ms; KO, n = 8; WT, n = 6; P < 0.05), which was eliminated with atropine. CaT duration at 80% and CaT alternans magnitude were similar between groups both with and without sympathetic nerve stimulation. These results indicate that cholinergic transdifferentiation following MI prolongs APD in the remote and border zone and reduces APD heterogeneity.NEW & NOTEWORTHY Cardiac sympathetic neurons undergo cholinergic transdifferentiation following myocardial infarction; however, the electrophysiological effects of corelease of norepinephrine and acetylcholine (ACh) have never been assessed. Using a mouse model in which choline acetyltransferase was deleted from adult noradrenergic neurons and optical mapping of innervated hearts, we found that corelease of ACh reduces dispersion of action potential duration, which may be antiarrhythmic.

Different paths, same destination: divergent action potential responses produce conserved cardiac fight-or-flight response in mouse and rabbit hearts.

KEY POINTS: Cardiac electrophysiology and Ca2+ handling change rapidly during the fight-or-flight response to meet physiological demands. Despite dramatic differences in cardiac electrophysiology, the cardiac fight-or-flight response is highly conserved across species. In this study, we performed physiological sympathetic nerve stimulation (SNS) while optically mapping cardiac action potentials and intracellular Ca2+ transients in innervated mouse and rabbit hearts. Despite similar heart rate and Ca2+ handling responses between mouse and rabbit hearts, we found notable species differences in spatio-temporal repolarization dynamics during SNS. Species-specific computational models revealed that these electrophysiological differences allowed for enhanced Ca2+ handling (i.e. enhanced inotropy) in each species, suggesting that electrophysiological responses are fine-tuned across species to produce optimal cardiac fight-or-flight responses. ABSTRACT: Sympathetic activation of the heart results in positive chronotropy and inotropy, which together rapidly increase cardiac output. The precise mechanisms that produce the electrophysiological and Ca2+ handling changes underlying chronotropic and inotropic responses have been studied in detail in isolated cardiac myocytes. However, few studies have examined the dynamic effects of physiological sympathetic nerve activation on cardiac action potentials (APs) and intracellular Ca2+ transients (CaTs) in the intact heart. Here, we performed bilateral sympathetic nerve stimulation (SNS) in fully innervated, Langendorff-perfused rabbit and mouse hearts. Dual optical mapping with voltage- and Ca2+ -sensitive dyes allowed for analysis of spatio-temporal AP and CaT dynamics. The rabbit heart responded to SNS with a monotonic increase in heart rate (HR), monotonic decreases in AP and CaT duration (APD, CaTD), and a monotonic increase in CaT amplitude. The mouse heart had similar HR and CaT responses; however, a pronounced biphasic APD response occurred, with initial prolongation (50.9 ± 5.1 ms at t = 0 s vs. 60.6 ± 4.1 ms at t = 15 s, P < 0.05) followed by shortening (46.5 ± 9.1 ms at t = 60 s, P = NS vs. t = 0). We determined the biphasic APD response in mouse was partly due to dynamic changes in HR during SNS and was exacerbated by ß-adrenergic activation. Simulations with species-specific cardiac models revealed that transient APD prolongation in mouse allowed for greater and more rapid CaT responses, suggesting more rapid increases in contractility; conversely, the rabbit heart requires APD shortening to produce optimal inotropic responses. Thus, while the cardiac fight-or-flight response is highly conserved between species, the underlying mechanisms orchestrating these effects differ significantly.

Calcium-Dependent Arrhythmogenic Foci Created by Weakly Coupled Myocytes in the Failing Heart.

RATIONALE: Intercellular uncoupling and Ca2+ (Ca) mishandling can initiate triggered ventricular arrhythmias. Spontaneous Ca release activates inward current which depolarizes membrane potential (Vm) and can trigger action potentials in isolated myocytes. However, cell-cell coupling in intact hearts limits local depolarization and may protect hearts from this arrhythmogenic mechanism. Traditional optical mapping lacks the spatial resolution to assess coupling of individual myocytes. OBJECTIVE: We investigate local intercellular coupling in Ca-induced depolarization in intact hearts, using confocal microscopy to measure local Vm and intracellular [Ca] simultaneously. METHODS AND RESULTS: We used isolated Langendorff-perfused hearts from control (CTL) and heart failure (HF) mice (HF induced by transaortic constriction). In CTL hearts, 1.4% of myocytes were poorly synchronized with neighboring cells and exhibited asynchronous (AS) Ca transients. These AS myocytes were much more frequent in HF (10.8% of myocytes, P<0.05 versus CTL). Local Ca waves depolarized Vm in HF but not CTL hearts, suggesting weaker gap junction coupling in HF-AS versus CTL-AS myocytes. Cell-cell coupling was assessed by calcein fluorescence recovery after photobleach during intracellular [Ca] recording. All regions in CTL hearts exhibited faster calcein diffusion than in HF, with HF-AS myocyte being slowest. In HF-AS, enhancing gap junction conductance (with rotigaptide) increased coupling and suppressed Vm depolarization during Ca waves. Conversely, in CTL hearts, gap junction inhibition (carbenoxolone) decreased coupling and allowed Ca wave-induced depolarizations. Synchronization of Ca wave initiation and triggered action potentials were observed in HF hearts and computational models. CONCLUSIONS: Well-coupled CTL myocytes are effectively voltage-clamped during Ca waves, protecting the heart from triggered arrhythmias. Spontaneous Ca waves are much more common in HF myocytes and these AS myocytes are also poorly coupled, enabling local Ca-induced inward current of sufficient source strength to overcome a weakened current sink to depolarize Vm and trigger action potentials.

Antiarrhythmic mechanisms of beta blocker therapy.

Sympathetic activity plays an important role in modulation of cardiac rhythm. Indeed, while exerting positive tropic effects in response to physiologic and pathologic stressors, ß-adrenergic stimulation influences cardiac electrophysiology and can lead to disturbances of the heart rhythm and potentially lethal arrhythmias, particularly in pathological settings. For this reason, ß-blockers are widely utilized clinically as antiarrhythmics. In this review, the molecular mechanisms of ß-adrenergic action in the heart, the cellular and tissue level cardiac responses to ß-adrenergic stimulation, and the clinical use of ß-blockers as antiarrhythmic agents are reviewed. We emphasize the complex interaction between cardiomyocyte signaling, contraction, and electrophysiology occurring over multiple time- and spatial-scales during pathophysiological responses to ß-adrenergic stimulation. An integrated understanding of this complex system is essential for optimizing therapies aimed at preventing arrhythmias.

Diabetic hyperglycaemia activates CaMKII and arrhythmias by O-linked glycosylation.

Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) is an enzyme with important regulatory functions in the heart and brain, and its chronic activation can be pathological. CaMKII activation is seen in heart failure, and can directly induce pathological changes in ion channels, Ca(2+) handling and gene transcription. Here, in human, rat and mouse, we identify a novel mechanism linking CaMKII and hyperglycaemic signalling in diabetes mellitus, which is a key risk factor for heart and neurodegenerative diseases. Acute hyperglycaemia causes covalent modification of CaMKII by O-linked N-acetylglucosamine (O-GlcNAc). O-GlcNAc modification of CaMKII at Ser 279 activates CaMKII autonomously, creating molecular memory even after Ca(2+) concentration declines. O-GlcNAc-modified CaMKII is increased in the heart and brain of diabetic humans and rats. In cardiomyocytes, increased glucose concentration significantly enhances CaMKII-dependent activation of spontaneous sarcoplasmic reticulum Ca(2+) release events that can contribute to cardiac mechanical dysfunction and arrhythmias. These effects were prevented by pharmacological inhibition of O-GlcNAc signalling or genetic ablation of CaMKIIδ. In intact perfused hearts, arrhythmias were aggravated by increased glucose concentration through O-GlcNAc- and CaMKII-dependent pathways. In diabetic animals, acute blockade of O-GlcNAc inhibited arrhythmogenesis. Thus, O-GlcNAc modification of CaMKII is a novel signalling event in pathways that may contribute critically to cardiac and neuronal pathophysiology in diabetes and other diseases.