Association of N-Acetyl Asparagine with QTc in Diabetes: A Metabolomics Study.

Changes in the cardio-metabolomics profile and hormonal status have been associated with long QT syndrome, sudden cardiac death and increased mortality. The mechanisms underlying QTc duration are not fully understood. Therefore, an identification of novel markers that complement the diagnosis in these patients is needed. In the present study, we performed untargeted metabolomics on the sera of diabetic patients at a high risk of cardiovascular disease, followed up for 2.55 [2.34-2.88] years (NCT02431234), with the aim of identifying the metabolomic changes associated with QTc. We used independent weighted gene correlation network analysis (WGCNA) to explore the association between metabolites clusters and QTc at T1 (baseline) and T2 (follow up). The overlap of the highly correlated modules at T1 and T2 identified N-Acetyl asparagine as the only metabolite in common, which was involved with the urea cycle and metabolism of arginine, proline, glutamate, aspartate and asparagine. This analysis was confirmed by applying mixed models, further highlighting its association with QTc. In the current study, we were able to identify a metabolite associated with QTc in diabetic patients at two chronological time points, suggesting a previously unrecognized potential role of N-Acetyl asparagine in diabetic patients suffering from long QTc.

Early Protective Role of Inflammation in Cardiac Remodeling and Heart Failure: Focus on TNFα and Resident Macrophages.

Cardiac hypertrophy, initiated by a variety of physiological or pathological stimuli (hemodynamic or hormonal stimulation or infarction), is a critical early adaptive compensatory response of the heart. The structural basis of the progression from compensated hypertrophy to pathological hypertrophy and heart failure is still largely unknown. In most cases, early activation of an inflammatory program reflects a reparative or protective response to other primary injurious processes. Later on, regardless of the underlying etiology, heart failure is always associated with both local and systemic activation of inflammatory signaling cascades. Cardiac macrophages are nodal regulators of inflammation. Resident macrophages mostly attenuate cardiac injury by secreting cytoprotective factors (cytokines, chemokines, and growth factors), scavenging damaged cells or mitochondrial debris, and regulating cardiac conduction, angiogenesis, lymphangiogenesis, and fibrosis. In contrast, excessive recruitment of monocyte-derived inflammatory macrophages largely contributes to the transition to heart failure. The current review examines the ambivalent role of inflammation (mainly TNFα-related) and cardiac macrophages (Mφ) in pathophysiologies from non-infarction origin, focusing on the protective signaling processes. Our objective is to illustrate how harnessing this knowledge could pave the way for innovative therapeutics in patients with heart failure.

Transcriptomic and Lipidomic Mapping of Macrophages in the Hub of Chronic Beta-Adrenergic-Stimulation Unravels Hypertrophy-, Proliferation-, and Lipid Metabolism-Related Genes as Novel Potential Markers of Early Hypertrophy or Heart Failure.

Sympathetic nervous system overdrive with chronic release of catecholamines is the most important neurohormonal mechanism activated to maintain cardiac output in response to heart stress. Beta-adrenergic signaling behaves first as a compensatory pathway improving cardiac contractility and maladaptive remodeling but becomes dysfunctional leading to pathological hypertrophy and heart failure (HF). Cardiac remodeling is a complex inflammatory syndrome where macrophages play a determinant role. This study aimed at characterizing the temporal transcriptomic evolution of cardiac macrophages in mice subjected to beta-adrenergic-stimulation using RNA sequencing. Owing to a comprehensive bibliographic analysis and complementary lipidomic experiments, this study deciphers typical gene profiles in early compensated hypertrophy (ECH) versus late dilated remodeling related to HF. We uncover cardiac hypertrophy- and proliferation-related transcription programs typical of ECH or HF macrophages and identify lipid metabolism-associated and Na+ or K+ channel-related genes as markers of ECH and HF macrophages, respectively. In addition, our results substantiate the key time-dependent role of inflammatory, metabolic, and functional gene regulation in macrophages during beta-adrenergic dependent remodeling. This study provides important and novel knowledge to better understand the prevalent key role of resident macrophages in response to chronically activated beta-adrenergic signaling, an effective diagnostic and therapeutic target in failing hearts.

Decrease of Pdzrn3 is required for heart maturation and protects against heart failure.

Heart failure is the final common stage of most cardiopathies. Cardiomyocytes (CM) connect with others via their extremities by intercalated disk protein complexes. This planar and directional organization of myocytes is crucial for mechanical coupling and anisotropic conduction of the electric signal in the heart. One of the hallmarks of heart failure is alterations in the contact sites between CM. Yet no factor on its own is known to coordinate CM polarized organization. We have previously shown that PDZRN3, an ubiquitine ligase E3 expressed in various tissues including the heart, mediates a branch of the Planar cell polarity (PCP) signaling involved in tissue patterning, instructing cell polarity and cell polar organization within a tissue. PDZRN3 is expressed in the embryonic mouse heart then its expression dropped significantly postnatally corresponding with heart maturation and CM polarized elongation. A moderate CM overexpression of Pdzrn3 (Pdzrn3 OE) during the first week of life, induced a severe eccentric hypertrophic phenotype with heart failure. In models of pressure-overload stress heart failure, CM-specific Pdzrn3 knockout showed complete protection against degradation of heart function. We reported that Pdzrn3 signaling induced PKC ζ expression, c-Jun nuclear translocation and a reduced nuclear ß catenin level, consistent markers of the planar non-canonical Wnt signaling in CM. We then show that subcellular localization (intercalated disk) of junction proteins as Cx43, ZO1 and Desmoglein 2 was altered in Pdzrn3 OE mice, which provides a molecular explanation for impaired CM polarization in these mice. Our results reveal a novel signaling pathway that controls a genetic program essential for heart maturation and maintenance of overall geometry, as well as the contractile function of CM, and implicates PDZRN3 as a potential therapeutic target for the prevention of human heart failure.

Epicardial origin of cardiac arrhythmias: clinical evidences and pathophysiology.

Recent developments in imaging, mapping, and ablation techniques have shown that the epicardial region of the heart is a key player in the occurrence of ventricular arrhythmic events in several cardiac diseases, such as Brugada syndrome, arrhythmogenic cardiomyopathy, or dilated cardiomyopathy. At the atrial level as well, the epicardial region has emerged as an important determinant of the substrate of atrial fibrillation, pointing to common underlying pathophysiological mechanisms. Alteration in the gradient of repolarization between myocardial layers favouring the occurrence of re-entry circuits has largely been described. The fibro-fatty infiltration of the subepicardium is another shared substrate between ventricular and atrial arrhythmias. Recent data have emphasized the role of the epicardial reactivation in the formation of this arrhythmogenic substrate. There are new evidences supporting this structural remodelling process to be regulated by the recruitment of epicardial progenitor cells that can differentiate into adipocytes or fibroblasts under various stimuli. In addition, immune-inflammatory processes can also contribute to fibrosis of the subepicardial layer. A better understanding of such 'electrical fragility' of the epicardial area will open perspectives for novel biomarkers and therapeutic strategies. In this review article, a pathophysiological scheme of epicardial-driven arrhythmias will be proposed.

Impacts of a high-fat diet on the metabolic profile and the phenotype of atrial myocardium in mice.

AIMS: Obesity, diabetes, and metabolic syndromes are risk factors of atrial fibrillation (AF). We tested the hypothesis that metabolic disorders have a direct impact on the atria favouring the formation of the substrate of AF. METHODS AND RESULTS: Untargeted metabolomic and lipidomic analysis was used to investigate the consequences of a prolonged high-fat diet (HFD) on mouse atria. Atrial properties were characterized by measuring mitochondria respiration in saponin-permeabilized trabeculae, by recording action potential (AP) with glass microelectrodes in trabeculae and ionic currents in myocytes using the perforated configuration of patch clamp technique and by several immuno-histological and biochemical approaches. After 16 weeks of HFD, obesogenic mice showed a vulnerability to AF. The atrial myocardium acquired an adipogenic and inflammatory phenotypes. Metabolomic and lipidomic analysis revealed a profound transformation of atrial energy metabolism with a predominance of long-chain lipid accumulation and beta-oxidation activation in the obese mice. Mitochondria respiration showed an increased use of palmitoyl-CoA as energy substrate. APs were short duration and sensitive to the K-ATP-dependent channel inhibitor, whereas K-ATP current was enhanced in isolated atrial myocytes of obese mouse. CONCLUSION: HFD transforms energy metabolism, causes fat accumulation, and induces electrical remodelling of the atrial myocardium of mice that become vulnerable to AF.

Remodeling of Ion Channel Trafficking and Cardiac Arrhythmias.

Both inherited and acquired cardiac arrhythmias are often associated with the abnormal functional expression of ion channels at the cellular level. The complex machinery that continuously traffics, anchors, organizes, and recycles ion channels at the plasma membrane of a cardiomyocyte appears to be a major source of channel dysfunction during cardiac arrhythmias. This has been well established with the discovery of mutations in the genes encoding several ion channels and ion channel partners during inherited cardiac arrhythmias. Fibrosis, altered myocyte contacts, and post-transcriptional protein changes are common factors that disorganize normal channel trafficking during acquired cardiac arrhythmias. Channel availability, described notably for hERG and KV1.5 channels, could be another potent arrhythmogenic mechanism. From this molecular knowledge on cardiac arrhythmias will emerge novel antiarrhythmic strategies.

Do Cellular Entry Mechanisms of SARS-Cov-2 Affect Myocardial Cells and Contribute to Cardiac Injury in COVID-19 Patients?

Although the main vital organ affected by SARS CoV-2 is the lung, more than 20% of hospitalized patients show heart injury, however, the underlying mechanisms are still actively investigated. Inflammation or myocardial ischemia are now well-established pathogenic factors. Direct cardiac damage by the virus is likely and might account for some aspects of cardiac disease in COVID-19 patients. However, precise knowledge on mechanisms of virus entry and progression in host cells and notably in cardiac cells is necessary in order to define the broad spectrum of pathogenicity of SARS-Cov-2 on myocardium and to identify specific therapeutic targets. This review will focus on the intracellular trafficking machinery, the Achilles heel of host cells, which can be used by the virus to infect cells of the cardiovascular system.

Microtubule polymerization state and clathrin-dependent internalization regulate dynamics of cardiac potassium channel: Microtubule and clathrin control of KV1.5 channel.

Ion channel trafficking powerfully influences cardiac electrical activity as it regulates the number of available channels at the plasma membrane. Studies have largely focused on identifying the molecular determinants of the trafficking of the atria-specific KV1.5 channel, the molecular basis of the ultra-rapid delayed rectifier current IKur. Besides, regulated KV1.5 channel recycling upon changes in homeostatic state and mechanical constraints in native cardiomyocytes has been well documented. Here, using cutting-edge imaging in live myocytes, we investigated the dynamics of this channel in the plasma membrane. We demonstrate that the clathrin pathway is a major regulator of the functional expression of KV1.5 channels in atrial myocytes, with the microtubule network as the prominent organizer of KV1.5 transport within the membrane. Both clathrin blockade and microtubule disruption result in channel clusterization with reduced membrane mobility and internalization, whereas disassembly of the actin cytoskeleton does not. Mobile KV1.5 channels are associated with the microtubule plus-end tracking protein EB1 whereas static KV1.5 clusters are associated with stable acetylated microtubules. In human biopsies from patients in atrial fibrillation associated with atrial remodeling, drastic modifications in the trafficking balance occurs together with alteration in microtubule polymerization state resulting in modest reduced endocytosis and increased recycling. Consequently, hallmark of atrial KV1.5 dynamics within the membrane is clathrin- and microtubule- dependent. During atrial remodeling, predominance of anterograde trafficking activity over retrograde trafficking could result in accumulation ok KV1.5 channels in the plasma membrane.

Distinct calcium/calmodulin-dependent serine protein kinase domains control cardiac sodium channel membrane expression and focal adhesion anchoring.

BACKGROUND: Membrane-associated guanylate kinase proteins function as adaptor proteins to mediate the recruitment and scaffolding of ion channels in the plasma membrane in various cell types. In the heart, the protein calcium/calmodulin-dependent serine protein kinase (CASK) negatively regulates the main cardiac sodium channel NaV1.5, which carries the sodium current (INa) by preventing its anterograde trafficking. CASK is also a new member of the dystrophin-glycoprotein complex and, like syntrophin, binds to the C-terminal domain of the channel. OBJECTIVE: The purpose of this study was to unravel the mechanisms of CASK-mediated negative INa regulation and interaction with the dystrophin-glycoprotein complex in cardiac myocytes. METHODS: CASK adenoviral truncated constructs with sequential single functional domain deletions were designed for overexpression in cardiac myocytes: CASKΔCAMKII, CASKΔL27A, CASKΔL27B, CASKΔPDZ, CASKΔSH3, CASKΔHOOK, and CASKΔGUK. A combination of whole-cell patch-clamp recording, total internal reflection fluorescence microscopy, and biochemistry experiments was conducted in cardiac myocytes to study the functional consequences of domain deletions. RESULTS: We show that both L27B and GUK domains are required for the negative regulatory effect of CASK on INa and NaV1.5 surface expression and that the HOOK domain is essential for interaction with the cell adhesion dystrophin-glycoprotein complex. CONCLUSION: This study demonstrates that the multimodular structure of CASK confers an ability to simultaneously interact with several targets within cardiomyocytes. Through its L27B, GUK, and HOOK domains, CASK potentially provides the ability to control channel delivery at adhesion points in cardiomyocytes.

Loss of spatacsin impairs cholesterol trafficking and calcium homeostasis.

Mutations in SPG11, leading to loss of spatacsin function, impair the formation of membrane tubules in lysosomes and cause lysosomal lipid accumulation. However, the full nature of lipids accumulating in lysosomes and the physiological consequences of such accumulation are unknown. Here we show that loss of spatacsin inhibits the formation of tubules on lysosomes and prevents the clearance of cholesterol from this subcellular compartment. Accumulation of cholesterol in lysosomes decreases cholesterol levels in the plasma membrane, enhancing the entry of extracellular calcium by store-operated calcium entry and increasing resting cytosolic calcium levels. Higher cytosolic calcium levels promote the nuclear translocation of the master regulator of lysosomes TFEB, preventing the formation of tubules and the clearance of cholesterol from lysosomes. Our work reveals a homeostatic balance between cholesterol trafficking and cytosolic calcium levels and shows that loss of spatacsin impairs this homeostatic equilibrium.

The Cardiac Sodium Channel and Its Protein Partners.

Activation of the electrical signal and its transmission as a depolarizing wave in the whole heart requires highly organized myocyte architecture and cell-cell contacts. In addition, complex trafficking and anchoring intracellular machineries regulate the proper surface expression of channels and their targeting to distinct membrane domains. An increasing list of proteins, lipids, and second messengers can contribute to the normal targeting of ion channels in cardiac myocytes. However, their precise roles in the electrophysiology of the heart are far from been extensively understood. Nowadays, much effort in the field focuses on understanding the mechanisms that regulate ion channel targeting to sarcolemma microdomains and their organization into macromolecular complexes. The purpose of the present section is to provide an overview of the characterized partners of the main cardiac sodium channel, NaV1.5, involved in regulating the functional expression of this channel both in terms of trafficking and targeting into microdomains.

Ion Channel Trafficking: Control of Ion Channel Density as a Target for Arrhythmias?

The shape of the cardiac action potential (AP) is determined by the contributions of numerous ion channels. Any dysfunction in the proper function or expression of these ion channels can result in a change in effective refractory period (ERP) and lead to arrhythmia. The processes underlying the correct targeting of ion channels to the plasma membrane are complex, and have not been fully characterized in cardiac myocytes. Emerging evidence highlights ion channel trafficking as a potential causative factor in certain acquired and inherited arrhythmias, and therapies which target trafficking as opposed to pore block are starting to receive attention. In this review we present the current evidence for the mechanisms which underlie precise control of cardiac ion channel trafficking and targeting.

cAMP-dependent regulation of IKs single-channel kinetics.

The delayed potassium rectifier current, IKs , is composed of KCNQ1 and KCNE1 subunits and plays an important role in cardiac action potential repolarization. During ß-adrenergic stimulation, 3'-5'-cyclic adenosine monophosphate (cAMP)-dependent protein kinase A (PKA) phosphorylates KCNQ1, producing an increase in IKs current and a shortening of the action potential. Here, using cell-attached macropatches and single-channel recordings, we investigate the microscopic mechanisms underlying the cAMP-dependent increase in IKs current. A membrane-permeable cAMP analog, 8-(4-chlorophenylthio)-cAMP (8-CPT-cAMP), causes a marked leftward shift of the conductance-voltage relation in macropatches, with or without an increase in current size. Single channels exhibit fewer silent sweeps, reduced first latency to opening (control, 1.61 ± 0.13 s; cAMP, 1.06 ± 0.11 s), and increased higher-subconductance-level occupancy in the presence of cAMP. The E160R/R237E and S209F KCNQ1 mutants, which show fixed and enhanced voltage sensor activation, respectively, largely abolish the effect of cAMP. The phosphomimetic KCNQ1 mutations, S27D and S27D/S92D, are much less and not at all responsive, respectively, to the effects of PKA phosphorylation (first latency of S27D + KCNE1 channels: control, 1.81 ± 0.1 s; 8-CPT-cAMP, 1.44 ± 0.1 s, P < 0.05; latency of S27D/S92D + KCNE1: control, 1.62 ± 0.1 s; cAMP, 1.43 ± 0.1 s, nonsignificant). Using total internal reflection fluorescence microscopy, we find no overall increase in surface expression of the channel during exposure to 8-CPT-cAMP. Our data suggest that the cAMP-dependent increase in IKs current is caused by an increase in the likelihood of channel opening, combined with faster openings and greater occupancy of higher subconductance levels, and is mediated by enhanced voltage sensor activation.

Lateral Membrane-Specific MAGUK CASK Down-Regulates NaV1.5 Channel in Cardiac Myocytes.

RATIONALE: Mechanisms underlying membrane protein localization are crucial in the proper function of cardiac myocytes. The main cardiac sodium channel, NaV1.5, carries the sodium current (INa) that provides a rapid depolarizing current during the upstroke of the action potential. Although enriched in the intercalated disc, NaV1.5 is present in different membrane domains in myocytes and interacts with several partners. OBJECTIVE: To test the hypothesis that the MAGUK (membrane-associated guanylate kinase) protein CASK (calcium/calmodulin-dependent serine protein kinase) interacts with and regulates NaV1.5 in cardiac myocytes. METHODS AND RESULTS: Immunostaining experiments showed that CASK localizes at lateral membranes of cardiac myocytes, in association with dystrophin. Whole-cell patch clamp showed that CASK-silencing increases INa in vitro. In vivo CASK knockdown similarly increased INa recorded in freshly isolated myocytes. Pull-down experiments revealed that CASK directly interacts with the C-terminus of NaV1.5. CASK silencing reduces syntrophin expression without affecting NaV1.5 and dystrophin expression levels. Total Internal Reflection Fluorescence microscopy and biotinylation assays showed that CASK silencing increased the surface expression of NaV1.5 without changing mRNA levels. Quantification of NaV1.5 expression at the lateral membrane and intercalated disc revealed that the lateral membrane pool only was increased upon CASK silencing. The protein transport inhibitor brefeldin-A prevented INa increase in CASK-silenced myocytes. During atrial dilation/remodeling, CASK expression was reduced but its localization remained unchanged. CONCLUSION: This study constitutes the first description of an unconventional MAGUK protein, CASK, which directly interacts with NaV1.5 channel and controls its surface expression at the lateral membrane by regulating ion channel trafficking.

Abnormal sodium current properties contribute to cardiac electrical and contractile dysfunction in a mouse model of myotonic dystrophy type 1.

Myotonic dystrophy type 1 (DM1) is the most common neuromuscular disorder and is associated with cardiac conduction defects. However, the mechanisms of cardiac arrhythmias in DM1 are unknown. We tested the hypothesis that abnormalities in the cardiac sodium current (INa) are involved, and used a transgenic mouse model reproducing the expression of triplet expansion observed in DM1 (DMSXL mouse). The injection of the class-I antiarrhythmic agent flecainide induced prominent conduction abnormalities and significantly lowered the radial tissular velocities and strain rate in DMSXL mice compared to WT. These abnormalities were more pronounced in 8-month-old mice than in 3-month-old mice. Ventricular action potentials recorded by standard glass microelectrode technique exhibited a lower maximum upstroke velocity [dV/dt](max) in DMSXL. This decreased [dV/dt](max) was associated with a 1.7 fold faster inactivation of INa in DMSXL myocytes measured by the whole-cell patch-clamp technique. Finally in the DMSXL mouse, no mutation in the Scn5a gene was detected and neither cardiac fibrosis nor abnormalities of expression of the sodium channel protein were observed. Therefore, alterations in the sodium current markedly contributed to electrical conduction block in DM1. This result should guide pharmaceutical and clinical research toward better therapy for the cardiac arrhythmias associated with DM1.

Cardiac-specific ablation of synapse-associated protein SAP97 in mice decreases potassium currents but not sodium current.

BACKGROUND: Membrane-associated guanylate kinase (MAGUK) proteins are important determinants of ion channel organization in the plasma membrane. In the heart, the MAGUK protein SAP97, encoded by the DLG1 gene, interacts with several ion channels via their PDZ domain-binding motif and regulates their function and localization. OBJECTIVE: The purpose of this study was to assess in vivo the role of SAP97 in the heart by generating a genetically modified mouse model in which SAP97 is suppressed exclusively in cardiomyocytes. METHODS: SAP97(fl/fl) mice were generated by inserting loxP sequences flanking exons 1-3 of the SAP97 gene. SAP97(fl/fl) mice were crossed with αMHC-Cre mice to generate αMHC-Cre/SAP97(fl/fl) mice, thus resulting in a cardiomyocyte-specific deletion of SAP97. Quantitative reverse transcriptase-polymerase chain reaction, western blots, and immunostaining were performed to measure mRNA and protein expression levels, and ion channel localization. The patch-clamp technique was used to record ion currents and action potentials. Echocardiography and surface ECGs were performed on anesthetized mice. RESULTS: Action potential duration was greatly prolonged in αMHC-Cre/SAP97(fl/fl) cardiomyocytes compared to SAP97(fl/fl) controls, but maximal upstroke velocity was unchanged. This was consistent with the decreases observed in IK1, Ito, and IKur potassium currents and the absence of effect on the sodium current INa. Surface ECG revealed an increased corrected QT interval in αMHC-Cre/SAP97(fl/fl) mice. CONCLUSION: These data suggest that ablation of SAP97 in the mouse heart mainly alters potassium channel function. Based on the important role of SAP97 in regulating the QT interval, DLG1 may be a susceptibility gene to be investigated in patients with congenital long QT syndrome.

Human epicardial adipose tissue induces fibrosis of the atrial myocardium through the secretion of adipo-fibrokines.

AIMS: Recent studies have reported a relationship between the abundance of epicardial adipose tissue (EAT) and the risk of cardiovascular diseases including atrial fibrillation (AF). However, the underlying mechanisms are unknown. The aim of this study was to examine the effects of the secretome of human EAT on the histological properties of the myocardium. METHODS AND RESULTS: Samples of EAT and subcutaneous adipose (SAT), obtained from 39 patients undergoing coronary bypass surgery, were analysed and tested in an organo-culture model of rat atria to evaluate the fibrotic properties of human fat depots. The EAT secretome induced global fibrosis (interstitial and peripheral) of rat atria in organo-culture conditions. Activin A was highly expressed in EAT compared with SAT and promoted atrial fibrosis, an effect blocked using neutralizing antibody. In addition, Activin A levels were enhanced in patients with low left-ventricular function. In sections of human atrial and ventricular myocardium, adipose and myocardial tissues were in close contact, together with fibrosis. CONCLUSION: This study provides the first evidence that the secretome from EAT promotes myocardial fibrosis through the secretion of adipo-fibrokines such as Activin A.