Murine Atrial Myocytes Research Articles

Hypoxia Promotes Atrial Tachyarrhythmias via Opening of ATP-Sensitive Potassium Channels.

Hypoxia-ischemia predisposes to atrial arrhythmia. Atrial ATP-sensitive potassium channel (KATP) modulation during hypoxia has not been explored. We investigated the effects of hypoxia on atrial electrophysiology in mice with global deletion of KATP pore-forming subunits. Whole heart KATP RNA expression was probed. Whole-cell KATP current and action potentials were recorded in isolated wild-type (WT), Kir6.1 global knockout (6.1-gKO), and Kir6.2 global knockout murine atrial myocytes. Langendorff-perfused hearts were assessed for atrial effective refractory period (ERP), conduction velocity, wavefront path length (WFPL), and arrhymogenicity under normoxia/hypoxia using a microelectrode array and programmed electrical stimulation. Heart histology was assessed. Expression patterns were essentially identical for all KATP subunit RNA across human heart, whereas in mouse, Kir6.1 and SUR2 (sulphonylurea receptor) were higher in ventricle than atrium, and Kir6.2 and SUR1 were higher in atrium. Compared with WT, Kir6.2 global knockout atrial myocytes had reduced tolbutamide-sensitive current and action potentials were more depolarized with slower upstroke and reduced peak amplitude. Action potential duration was prolonged in 6.1-gKO atrial myocytes, absent of changes in other ion channel gene expression or atrial myocyte hypertrophy. In Langendorff-perfused hearts, baseline atrial ERP was prolonged and conduction velocity reduced in both KATP knockout mice compared with WT, without histological fibrosis. Compared with baseline, hypoxia led to conduction velocity slowing, stable ERP, and WFPL shortening in WT and 6.1-gKO hearts, whereas WFPL was stable in Kir6.2 global knockout hearts due to ERP prolongation with conduction velocity slowing. Tolbutamide reversed hypoxia-induced WFPL shortening in WT and 6.1-gKO hearts through ERP prolongation. Atrial tachyarrhythmias inducible with programmed electrical stimulation during hypoxia in WT and 6.1-gKO mice correlated with WFPL shortening. Spontaneous arrhythmia was not seen. KATP block/absence leads to cellular and tissue level atrial electrophysiological modification. Kir6.2 global knockout prevents hypoxia-induced atrial WFPL shortening and atrial arrhythmogenicity to programmed electrical stimulation. This mechanism could be explored translationally to treat ischemically driven atrial arrhythmia.

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.

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.

Characterization of Intracellular Sodium Homeostasis in Murine Atrial Myocytes

Intracellular sodium concentration ([Na+]i) is an important regulator of intracellular Ca2+ and provides insight into the activation of the sarcolemmal sodium calcium exchanger (NCX) and the behavior of both Na+ channels and the Na+,K+-ATPase. In atrial diseases such as atrial fibrillation (AF), intracellular Ca2+ signaling is profoundly altered. While the mechanisms underlying altered intracellular Ca2+ homeostasis are well characterized, the role that [Na+]i may play and its dysregulation in disease in atrial myocytes is less well understood. For example, we have previously shown that high rate (10 Hz) atrial activation for 5 days in a rabbit model leads to an expected but significant reduction in [Na+]i (Greiser et al, JCI, 2014; 124: 4759-72).Here we characterize intracellular Na+ homeostasis in murine atrial myocytes using the Na+ indicator SBFI (Molecular Probes) in quiescent cells. Dual UV excitation (340 nm and 380 nm) was provided by a rapid switching Sutter DG5-plus illuminator using a 300 W xenon light source with widefield imaging by an EMCCD camera (Princeton Instruments). Freshly isolated atrial myocytes were loaded with 10 μmol/l SBFI-AM for 90 min in the presence of the non-ionic surfactant Pluronic F-127 (0.05 %w/v). After washout of the extra cellular dye, SBFI-AM de-esterification was allowed for 20 minutes before proceeding with [Na+]i measurements. Fluorescence images (F340 and F380) were collected at 510 ± 40 nm. The calibration of [Na+]i was performed in each cell separately after the measurements of quiescent [Na+]i by exposing the SBFI-loaded myocytes to various extracellular [Na+] ([Na+]o) in the presence of 10 μmol/l gramicidin D and 100 μmol/l-strophanthidin. Resting [Na+]i in mouse atrial myocytes was 8.2 ± 0.6 mM. External field stimulation (4 ms, 20 V, 1 Hz) increased [Na+]i by 3.1 ± 0.2 mM. These data show that [Na+]i in murine atrial myocytes is lower than in murine ventricular myocytes (12 ± 1 mM, Correll et al, Circ Res, 2014;114:249-56). These data and more will provide a broad understanding of atrial Na+ homeostasis and its implication for atrial myocyte Ca2+ signaling.

Stretch-Induced Changes in Atrial Ca Signaling

Atrial myocytes undergo stretch during diastole as do ventricular myocytes. We have identified a novel mechanism that links cellular stretch in ventricular myocytes to the tuning of Ca2+ release from the sarcoplasmic reticulum (SR)(Prosser et al. Science 2011;333:1440-5). This mechanism, “X-ROS signaling”, depends on NOX2 (NADPH oxidase) to generate reactive oxygen species (ROS), which appears to oxidize ryanodine receptors (SR Ca2+ release channels) and increases their sensitivity to [Ca2+]i. Stretch mediates X-ROS signaling through microtubules, which appear to interact with NOX2 to enable it to generate ROS.We evaluated X-ROS signaling in murine atrial myocytes (C57/B6). Ca2+ sparks were recorded before and after stretch (10-20% of cell length). Ca2+ spark frequency increased during stretch and returned to pre-stretch values during relaxation. Interestingly, microtubule density was higher and protein expression levels were increased in murine atria compared to ventricles (Fig 1 A). Similarly, protein expression levels of the catalytic NOX2 subunit gp91phox were higher in atria compared to ventricles (Fig 1 A). Inhibition of gp91phox with the inhibitory peptide gp91ds-tat prevented stretch-induced Ca2+ spark increases in atrial myocytes (Fig 1 B). Thus, X-ROS signaling is also operative in atrial myocytes.

D: A DYSTROPHIN-DEPENDENT LOSS OF NEURONAL NITRIC OXIDE SYNTHASE IN ATRIAL FIBRILLATION (AF) PROMOTES ELECTRICALREMODELLING AND CREATES A SUBSTRATE FOR THE MAINTENANCE OF AF

Rationale Nitric oxide production in the heart is involved in the regulation of ion channels activity, myocardial perfusion and thrombogenesis. Atrial fibrillation (AF) has been associated with reduced myocardial nitric oxide (NO) bioavailability; however, the mechanisms and the consequences of this phenomenon remain to be elucidated. Methods and Results We evaluated the activity, protein content, and localization of constitutive NO synthases (NOS) in atrial tissue from 62 patients with permanent AF and 148 controls in normal sinus rhythm (SR), and in goats after 2 weeks (2W-AF, n=15) or 6 months (6M-AF, n=10) of pacing-induced AF ( vs. sham surgery & SR, n=14). NOS activity was uncoupled in right atrial (RA) tissue from AF patients and 6M-AF goats, both of which also exhibited an ipsilateral reduction in the NOS cofactor tetrahydrobiopterin (BH4) and an increase in arginase activity (HPLC). Although ex vivo BH4 and arginine supplementation reversed NOS uncoupling, atrial NOS activity remained profoundly suppressed in AF due to a progressive decrease in the “neuronal” isoform of NOS (nNOS) in atrial cardiomyocytes (by 65% in 2W-AF, 86% in 6M-AF goats and 62% in patients with AF). Atrial nNOS mRNA expression (qRT-PCR) did not differ between AF and SR; however, nNOS ubiquitination was increased in AF. Atrial nNOS protein content was partially restored by inhibition of proteasomal activity with MG132 but not by inhibiting the autophagy-lysosomal pathway with bafilomycin A1 or by pre-treatment with BH4. nNOS signalling and protein stability in the cardiac muscle depends on the enzyme9s anchoring to the dystrophin-syntrophin complex at the sarcolemmal membrane. In the fibrillating atrial myocardium we observed a significant reduction in dystrophin (by 56%) and its associated proteins, 1α syntrophin and caveolin-3; by contrast no difference in the dystrophin complex was observed in the atrial myocardium of nNOS -/- mice. Immunoprecipitation showed co-localisation of nNOS with dystrophin in SR, but not in AF. To assess whether loss of nNOS and reduced NO bioavailability played a role in the AF-induced atrial electrical remodelling; we evaluated the effect of nNOS inhibition or gene deletion in human and murine atrial myocytes. In RA myocytes from patients in SR (but not in patients with AF), nNOS inhibition shortened the action potential duration (APD) by 40%, and decreased the rate dependent-adaptation of APD (both of which are a hallmark of AF-induced electrical remodelling). These findings were recapitulated in murine right atrial myocytes in the presence of nNOS gene deletion or inhibition. In mice, nNOS gene deletion was sufficient to induce a 2-fold increase in the likelihood of developing AF in response to electrical stimulation. Conclusions - These data indicate that dystrophin-dependent loss of myocardial nNOS in AF is sufficient to produce an atrial electrical substrate that increases both inducibility and maintenance of this arrhythmia. Therapeutic strategies aimed at preserving or restoring the dystrophin complex may have a profound effect on NO bioavailability and the electrical substrate of AF.

Atrial Specific Stretch-Dependent Sub-Cellular Ca2+ Signaling

Atrial myocytes undergo stretch during diastole as do ventricular myocytes. We have recently identified a novel mechanism that links cellular stretch in ventricular myocytes to the tuning of Ca2+ release from the sarcoplasmic reticulum (SR)(Prosser et al. Science 2011;333:1440-5). This mechanism, “X-ROS signaling”, depends on NOX2 (NADPH oxidase) in the sarcolemmal and transverse tubule (TT) membranes to generate reactive oxygen species (ROS), which appears to oxidize the nearby (nanometers) ryanodine receptors (SR Ca2+ release channels) and increases their sensitivity to [Ca2+]i. Stretch mediates X-ROS signaling through microtubules, which appear to interact with NOX2 to enable it to generate ROS. We evaluated the effect of acute and repeated stretch on sub-cellular Ca2+ sparks in murine atrial myocytes (C57/B6; Figure 1A). Ca2+ sparks were recorded before and after stretch (8% of cell length). Ca2+ spark frequency increased during stretch and returned to pre-stretch values during relaxation (Figure 1B). Thus, X-ROS signaling and the consequent stretch-dependent Ca2+ release “tuning” found in ventricular myocytes is also operative in atrial myocytes. We will present data on the importance of NOX2, TTs and other components of the X-ROS signaling pathway in health and disease in atrial myocytes.

Stretch-Dependent Sub-Cellular Ca2+ Signaling in Atrial Myocytes

Atrial fibrillation (AF) is a multifactorial disease whose underlying mechanisms are still incompletely understood. Atrial myocytes undergo similar stretch during diastolic filling as ventricular myocytes and acute atrial stretch can induce AF. Our group recently identified a novel mechanism linking cellular stretch to the tuning of Ca2+ release from the sarcoplasmic reticulum (SR) in ventricular myocytes (Prosser et al. Science 201;333:1440-5). This mechanism, “X-ROS signaling”, depends on NOX2 (NADPH oxidase) to generate reactive oxygen species (ROS), which oxidizes the local RyR2 Ca2+ release channels of the SR and increases their Ca2+ sensitivity. Stretch mediates X-ROS signaling through microtubules, which interact with NOX2 located in transverse tubules and the surface sarcolemmal membranes.Interestingly, in atrial myocytes from AF patients both increased ROS levels and arrhythmogenic Ca2+ signaling instability are present. We evaluated the effect of acute and repeated stretch on sub-cellular Ca2+ sparks in murine atrial myocytes (C57/B6) with instrumentation (WPI, Sarasota, FL) and methods of Prosser et al. (2011). Ca2+ sparks were recorded before and after acute and repeated stretch (8% of cell length). Here, we report an increase in stretch-dependent Ca2+ spark frequency in single murine atrial myocytes. Thus, stretch-dependent Ca2+ release mechanisms are also operative in atrial myocytes and likely to play a role in Ca2+ dependent arrhythmias like AF. Our ongoing work will interrogate the components of the X-ROS pathway to determine the relative contribution of this pathway in atrial myocytes.

Molecular characterization of thyroid hormone-inhibited atrial L-type calcium channel expression: implication for atrial fibrillation in hyperthyroidism

Atrial fibrillation (AF) is a common complication in hyperthyroidism. Earlier studies demonstrate that thyroid hormone decreases L-type calcium channel (LCC) current expression with resultant shortening of action potential duration (APD), providing a substrate for AF. The aim of this study was to investigate the potential mechanism underlying the regulatory effect of thyroid hormone on LCC. In a hyperthyroid rat model, thyroid hormone (triiodothyronine [T3]) administration down-regulated atrial LCC expression. In vitro, treatment of murine atrial myocytes (HL-1) with T3 decreased the expression of LCC and its current, resulting in abbreviation of APD. Furthermore, T3 inhibited the activation of cyclic AMP response element (CRE)-binding protein (CREB), including phosphorylation at Ser133 and its nuclear translocation. Transient transfection studies in HL-1 cells indicated that T3 reduced LCC promoter activity. Deletion and mutation analysis of the LCC promoter region along with chromatin immunoprecipitation using anti-CREB antibody showed that CRE was essential for T3-mediated LCC gene expression. Transfection of dominant-negative CREB (mutated Ser133) and mutant thyroid hormone receptor (TR, mutated Cys51) abolished the T3-dependent effects, suggesting an association between both transcriptional factors. Co-immunoprecipitation documented an increased binding of TR with CREB after T3 treatment. The transcriptional cross-talk 3 between TR and CREB bound to CRE mediates T3-inhibited CREB activity and LCC expression. Thyroid hormone-induced TR binding of CREB inhibits CREB activity and LCC current expression, which may contribute to AF. These findings provide an important mechanistic insight into hyperthyroidism-induced AF.

Extracellular ATP Evokes Infiltration of Macrophage in Stretched Atrium

Introduction:Atrial inflammation and dilatation are risk factors for atrial fibrillation. We aimed to clarify the mechanism of macrophage infiltration in stretched atrium.Methods:In vitro studies were performed using murine atrial myocytes (HL-1). Murine macrophages were co-cultured with mechanically stretched HL-1 dells using modified Boyden chamber, with or without drugs including carbenoxolone (CBX, a gap junction channel blocker), apyrase, and pyridoxalphosphate-6-azophenyl-2′,4′-disulphonic acid (PPADS). Extracellular ATP concentration was measured under mechanically stretched HL-1.In in vivo study, mice underwent transverse aortic constriction (TAC) to induce atrial stretch, with or without CBX administration. Mice were sacrificed 10 days after the surgery for histological evaluation. Electrophysiological study including induction of atrial tachyarrhythmias (AT) was done in Langendorff-perfused heart.Results:Migration of macrophage was significantly increased from control to stretched condition, which was inhibited by CBX, apyrase, and PPADS. Extracellular ATP was increased from control to stretched condition, which was also inhibited by CBX. TAC induced infiltration of macrophage and deposition of collagen fiber in atrium. Inducibility of AT was significantly higher in TAC operated mice. These changes were inhibited by CBX administration.Conclusions:ATP released from stretched myocytes via gap junction channel is involved in macrophage recruitment and atrial arrhytmogenecity.

Connexin40 and connexin43 determine gating properties of atrial gap junction channels

While ventricular gap junctions contain only Cx43, atrial gap junctions contain both Cx40 and Cx43; yet the functional consequences of this co-expression remain poorly understood. We quantitated the expression of Cx40 and Cx43 and their contributions to atrial gap junctional conductance ( g j). Neonatal murine atrial myocytes showed similar abundances of Cx40 and Cx43 proteins, while ventricular myocytes contained at least 20 times more Cx43 than Cx40. Since Cx40 gap junction channels are blocked by 2 mM spermine while Cx43 channels are unaffected, we used spermine block as a functional dual whole cell patch clamp assay to determine Cx40 contributions to cardiac g j. Slightly more than half of atrial g j and ≤ 20% of ventricular g j were inhibited. In myocytes from Cx40 null mice, the inhibition of ventricular g j was completely abolished, and the block of atrial g j was reduced to < 20%. Compared to ventricular gap junctions, the transjunctional voltage ( V j)-dependent inactivation of atrial g j was reduced and kinetically slowed, while the V j-dependence of fast and slow inactivation was unchanged. We conclude that Cx40 and Cx43 are equally abundant in atrium and make similar contributions to atrial g j. Co-expression of Cx40 accounts for most, but not all, of the differences in the V j-dependent gating properties between atrium and ventricle that may play a role in the genesis of slow myocardial conduction and arrhythmias.

In Vivo Effect of a Dominant Negative Kv4.2 Loss-of-Function Mutation Eliminating Ito,f on Atrial Refractoriness and Atrial Fibrillation in Mice

Gain-of-function K(+) channel mutations cause familial atrial fibrillation (AF) by shortening of the atrial action potential duration (APD). APD-prolonging K(+) channel blockers are an effective therapeutic option in AF. In vitro, the dominant negative Kv4.2W362F mutation (Kv4DN) eliminates I(to,f) in murine atrial myocytes and markedly prolongs the APD, so whether this loss-of-function of I(to,f) alters the atrial effective refractory period (AERP) in vivo and/or affects AF-inducibility was investigated in the present study. Transvenous electrophysiological studies were performed in vivo in Kv4DN and wild-type littermate control (LMC) mice. Intriguingly, no difference was found between Kv4DN and LMC for the AERP in vivo either at baseline or after carbachol. Consequently, AF-inducibility at baseline (Kv4DN: 10/16 vs LMC: 7/13) and after carbachol (Kv4DN: 9/16 vs LMC: 6/13) did not differ between groups. However, AF-inducibility was associated with a significantly shorter AERP (inducible 51.1 +/-1.4 vs non-inducible 58.4 +/-1.6; P<0.01) irrespective of genotype. The loss-of-function of I(to,f) prolongs the APD in mouse atrial myocytes in vitro, but this effect on single cells does not translate into measurable AERP prolongation in vivo and hence does not exert an anti-arrhythmic effect. However, the susceptibility of mice to AF in vivo is determined by the individual AERP, irrespective of genotype.

Identification of a T-Type Ca 2+ Channel Isoform in Murine Atrial Myocytes (AT-1 Cells)

Calcium channels are important targets for therapeutics, but their molecular diversity complicates characterization of these channels in native heart cells. In this study, we identify a new splice variant of a low-voltage activated, or T-type Ca(2+), channel in murine atrial myocytes. To date, alpha1G and alpha1H are the only 2 T-type Ca(2+) channel isoforms found in cardiovascular tissue. We compared alpha1G and alpha1H channel current heterologously expressed in HEK 293 cells with T-type current from the murine atrial tumor cell, AT-1. AT-1 cell T-type current (I(T)) has the same voltage dependence of activation and inactivation as alpha1G and alpha1H. The cloned T-type channels and AT-1 T-type current share similar kinetics of macroscopic inactivation and deactivation. The kinetics of recovery from inactivation of T-type currents serves as an electrophysiological signature for T-channel isoform. alpha1G and AT-1 I(T) have a similar recovery from inactivation time course that is faster than that for alpha1H. In all cases, T-type current recovers with a biexponential time course, and the relative amplitude of fast and slow time courses explains the slower alpha1H recovery kinetics, rather than differences in the time constants of the individual transitions. Thus, the T-type channels may be an important contributor to automaticity in heart cells, and molecular diversity is reflected in the pathway of recovery from inactivation.