Adult Human Atria Research Articles

Human induced pluripotent stem cell-derived atrial cardiomyocytes recapitulate contribution of the slowly activating delayed rectifier currents IKs to repolarization in the human atrium.

Human induced pluripotent stem cell-derived atrial cardiomyocytes (hiPSC-aCM) could be a helpful tool to study the physiology and diseases of the human atrium. To fulfil this expectation, the electrophysiology of hiPSC-aCM should closely resemble the situation in the human atrium. Data on the contribution of the slowly activating delayed rectifier currents (IKs) to repolarization are lacking for both human atrium and hiPSC-aCM. Human atrial tissues were obtained from patients with sinus rhythm (SR) or atrial fibrillation (AF). Currents were measured in human atrial cardiomyocytes (aCM) and compared with hiPSC-aCM and used to model IKs contribution to action potential (AP) shape. Action potential was recorded by sharp microelectrodes. HMR-1556 (1 µM) was used to identify IKs and to estimate IKs contribution to repolarization. Less than 50% of hiPSC-aCM and aCM possessed IKs. Frequency of occurrence, current densities, activation/deactivation kinetics, and voltage dependency of IKs did not differ significantly between hiPSC-aCM and aCM, neither in SR nor AF. β-Adrenoceptor stimulation with isoprenaline did not increase IKs neither in aCM nor in hiPSC-aCM. In tissue from SR, block of IKs with HMR-1556 did not lengthen the action potential duration, even when repolarization reserve was reduced by block of the ultra-rapid repolarizing current with 4-aminopyridine or the rapidly activating delayed rectifier potassium outward current with E-4031. I Ks exists in hiPSC-aCM with biophysics not different from aCM. As in adult human atrium (SR and AF), IKs does not appear to relevantly contribute to repolarization in hiPSC-aCM.

A critical role of retinoic acid concentration for the induction of a fully human-like atrial action potential phenotype in hiPSC-CM

Retinoic acid (RA) induces an atrial phenotype in human induced pluripotent stem cells (hiPSCs), but expression of atrium-selective currents such as the ultrarapid (IKur) and acetylcholine-stimulated K+ current is variable and less than in the adult human atrium. We suspected methodological issues and systematically investigated the concentration dependency of RA. RA treatment increased IKur concentration dependently from 1.1± 0.54pA/pF (0 RA) to 3.8± 1.1, 5.8± 2.5, and 12.2± 4.3 at 0.01, 0.1, and 1μM, respectively. Only 1μM RA induced enough IKur to fully reproduce human atrial action potential (AP) shape and a robust shortening of APs upon carbachol. We found that sterile filtration caused substantial loss of RA. We conclude that 1μM RA seems to be necessary and sufficient to induce a full atrial AP shape in hiPSC-CM in EHT format. RA concentrations are prone to methodological issues and may profoundly impact the success of atrial differentiation.

Bile acids induce arrhythmias in human atrial myocardium—implications for altered serum bile acid composition in patients with atrial fibrillation

ObjectiveHigh bile acid serum concentrations have been implicated in cardiac disease, particularly in arrhythmias. Most data originate from in vitro studies and animal models. We tested the hypotheses that (1)...

A novel adult human atrium slice technique for studying electrical remodeling in atrial fibrillation

A novel adult human atrium slice technique for studying electrical remodeling in atrial fibrillation

Abstract 18084: Systematic Analysis of Gene Expression Differences Between Left and Right Atria in Different Mouse Strains and in Human Atrial Tissue

Background: Normal development of the atria requires left-right differentiation during embryonic development. Reduced expression of Pitx2c (Paired-like homeodomain transcription factor2, isoform c), a key regulator of left-right asymmetry, has recently been linked to atrial fibrillation. To characterize differences between left and right atrium in adult hearts, we systematically studied the molecular composition of left and right atrial tissue in adult murine and human atria. Methods: We compared left and right atrial gene expression in healthy, adult mice of different strains (MF1, 3 months old; MF1, 12 months old; Swiss Agouti, 12 months old) by employing whole genome array analyses on freshly frozen atrial tissue. Selected genes with enriched expression in right or left atrium were validated by RT-qPCR in further animals, and by RT-qPCR and Western blot in shock-frozen left and right atrial appendages of patients undergoing open heart surgery. Results: We identified 77 genes with preferential expression in one atrium that were common in all strains and age groups analysed. Independent of strain and age, Pitx2c was the gene with the highest enrichment in left atrium, while Bmp10 (Bone morphogenetic protein 10) showed enrichment in right atrium, consistent with prior reports. Nine selected genes, including Pitx2c, were validated by RT-qPCR in murine tissue, and five orthologous genes in human tissue. Western blot showed a 2-fold left-right ratio in PITX2c protein in adult human atria. Several of the genes and gene groups enriched in left atria have a reasonable biological role for the prevention of atrial pathology including atrial fibrillation, membrane electrophysiology, metabolic cellular function, and regulation of inflammatory cells. Comparison of the array data sets with published array analyses in heterozygous pitx2+/- atria suggested that approximately half of the genes with left-sided enrichment are regulated by Pitx2c. Conclusions: Our study reveals systematic differences between left and right atrial gene expression during adulthood and supports the hypothesis that Pitx2c has a functional role in maintaining “leftness” in the atrium in adult mice and humans.

Systematic analysis of gene expression differences between left and right atria in different mouse strains and in human atrial tissue.

BackgroundNormal development of the atria requires left-right differentiation during embryonic development. Reduced expression of Pitx2c (paired-like homeodomain transcription factor 2, isoform c), a key regulator of left-right asymmetry, has recently been linked to atrial fibrillation. We therefore systematically studied the molecular composition of left and right atrial tissue in adult murine and human atria.MethodsWe compared left and right atrial gene expression in healthy, adult mice of different strains and ages by employing whole genome array analyses on freshly frozen atrial tissue. Selected genes with enriched expression in either atrium were validated by RT-qPCR and Western blot in further animals and in shock-frozen left and right atrial appendages of patients undergoing open heart surgery.ResultsWe identified 77 genes with preferential expression in one atrium that were common in all strains and age groups analysed. Independent of strain and age, Pitx2c was the gene with the highest enrichment in left atrium, while Bmp10, a member of the TGFβ family, showed highest enrichment in right atrium. These differences were validated by RT-qPCR in murine and human tissue. Western blot showed a 2-fold left-right concentration gradient in PITX2 protein in adult human atria. Several of the genes and gene groups enriched in left atria have a known biological role for maintenance of healthy physiology, specifically the prevention of atrial pathologies involved in atrial fibrillation, including membrane electrophysiology, metabolic cellular function, and regulation of inflammatory processes. Comparison of the array datasets with published array analyses in heterozygous Pitx2c+/− atria suggested that approximately half of the genes with left-sided enrichment are regulated by Pitx2c.ConclusionsOur study reveals systematic differences between left and right atrial gene expression and supports the hypothesis that Pitx2c has a functional role in maintaining “leftness” in the atrium in adult murine and human hearts.

HNK-1 expression patterns in the embryonic rat heart distinguish between sinuatrial tissues and atrial myocardium.

HNK-1 expression was studied by immunohistochemistry in serial sections of embryonic and fetal rat hearts from 11.5 to 16.5 embryonic days. Graphic reconstructions were made to obtain detailed 3D information on the localization of immunoreactive tissues. The antibody used appeared to stain most parts of the venous sinus and the sinuatrial transitional zone as well as the atrioventricular transitional zone, but the patterns varied through the different developmental stages. At 11.5 days, positive myocardium was found in the right atrium and on top of the ventricular septal primordium. At 13.5 days, the left venous valve and the posterior atrial wall containing the orifice of the pulmonary vein were immunoreactive, and so were the right venous valve, the septum spurium and the superior, right-lateral and inferior parts of the atrioventricular canal. From the latter, immunoreactivity continued onto the crest of the ventricular septum. At 15.5 days, HNK-1 positivity in the two venous valves had become continuous, whereas the right-lateral part of the atrioventricular canal had lost its positivity, thus making the positive areas in the superior and inferior parts of this canal discontinuous. From the venous valves immunoreaction continued into the venous sinus septum but this area remained discontinuous with the inferior part of the atrioventricular canal. It is concluded that the entirety of venous sinus and sinuatrial transitional zone expresses the HNK-1 antigen and that the orifice of the pulmonary vein belongs to this complex, rather than to the embryonic atrium proper, which is HNK-1 negative. Extrapolation of these data to the adult human atrium leads to the conclusion hat most "atrial septal structures" are of sinuatrial origin, leaving the flap valve of the oval fossa (atrial septum primum) as the only really atrial structure. It is suggested that the atrioventricular node is derived from the inferior portion of the atrioventricular canal, and that two expansions of sinuatrial tissue form the substrate for anterior and posterior atrionodal inputs which in the literature have been described as internodal tracts.

Developmental changes of the ultrarapid delayed rectifier K + current in rat ventricular myocytes

Recent studies have shown the presence of a 4-aminopyridine (4-AP) sensitive, ultrarapid delayed rectifier K+ current (IK,ur) in adult human atria, but an apparent absence of this current in adult human ventricles. The present experiment was designed to investigate the postnatal changes of IK,ur in rat ventricular myocytes. The presence of IK,ur was evaluated with the use of a low concentration of 4-AP (50 micro;M). In 3-day-old newborns, the channel activity of rapidly activating outward current was predominantly the transient outward current (Ito). IK,ur could be recorded in a small number of 3-day-old cells lacking Ito (16%). In 10-day-old and adult rat ventricular myocytes, almost all cells expressed Ito and its current density increased significantly with age. The fraction of cells expressing IK,ur dramatically decreased with age and no IK,ur-like component could be detected in the adult cells. These findings first demonstrate that the expression of IK,ur is regulated developmentally in mammalian ventricular myocytes.

L-Type Calcium Current in Pediatric and Adult Human Atrial Myocytes: Evidence for Developmental Changes in Channel Inactivation

Animal studies have documented the presence of marked, species-dependent, developmental changes in the properties of the L-type calcium current in cardiac myocytes. In an effort to understand the postnatal changes which occur in the calcium current in human heart, we characterized the calcium current in atrial myocytes isolated from 17 pediatric and older children (ages 3 d to 14 y) and 12 adult (ages 43-79 y) human hearts using the whole-cell patch clamp technique. In contrast to animal models, we found no evidence for age-related changes in calcium current density, steady-state inactivation, or kinetics of recovery from inactivation, suggesting that, in human atrium, calcium channels are in many aspects functionally mature at the time of birth. However, statistically significant differences were found in the kinetics of calcium current inactivation, with calcium current measured in cells isolated from pediatric human atria inactivating approximately 2-fold faster than cells isolated from adult hearts. These results suggest a possible role for age-related changes in calcium current inactivation in the shortened action potential duration observed in pediatric compared with adult human atrium.

Augmented Expression of Atrial Myosin Light Chain 1 in Ventricular Aneurysms of Human: Enzyme Immunoassay for Atrial Myosin Light Chain 1

We established an enzyme immunoassay (EIA) for atrial myosin light chain 1 (ALC1) using monoclonal antibodies KA1 and KB1, which were specific for ALC1 and for both ALC1 and ventricular myosin light chain 1, respectively. The serum ALC1 levels of healthy subjects were 0.28 ± 0.14 ng/ml (mean ± SD). The tissue ALC1 levels of normal adult human atria were much higher than those of ventricles (p<0.01, 2,120 ± 1,200 in right atria, 2,180 ± 1,450 in left atria vs. 36.0 ± 20.2 in right ventricles, 37.7 ± 15.3 in left ventricles, ng/mg of proteins). The tissue ALC1 levels of ventricular aneurysms were significantly higher than those of normal ventricles (p<0.01, 206.7 ± 101.8). These results indicate that ALC1 is augmented in aneurysms and that the EIA provides a useful tool to investigate the roles of ALC1.

Novel monoclonal antibodies specific for human cardiac myosin light-chain 1: useful tools for analysis of normal and pathological hearts.

To investigate the developmental, physiological and pathophysiological roles of human cardiac myosin light-chain 1 (LC1s), we developed two novel monoclonal antibodies (KA1 and KB1) against human cardiac LC1s and examined LC1s in normal and pathological hearts immunohistochemically. KA1 and KB1 were specific only for atrial LC1 (ALC1) and for both ALC1 and ventricular LC1 (VLC1), respectively, in human hearts. Among human tissues tested, including skeletal muscle, vascular smooth muscle, and liver, KA1 did not crossreact with proteins in any other tissues than atria, whereas KB1 crossreacted with the slow-type LC1 of skeletal muscle. Among adult mammalian hearts of several other species including pig, dog, hamster, and rat, KA1 and KB1 crossreacted only with ALC1 and with both ALC1 and VLC1, respectively. ALC1 was strongly and uniformly observed in human fetal atria and ventricles and in normal adult human atria, but sporadically in normal adult human ventricles. In the overloaded ventricle (dilated cardiomyopathy), ALC1 was highly augmented but not uniform. These results suggest that the fetal VLC1 is immunohistochemically identical to the adult type of ALC1 and that ALC1 is expressed homogeneously in human fetal ventricles and sporadically in normal adult ventricles, and is re-expressed heterogeneously and in an increased amount in the overloaded ventricle.

Properties and modulation of Ca channels in adult human atrial cells

Ca-channel currents have been investigated in single cells isolated from adult human atrium using the whole-cell patch clamp technique. Ca-channel currents are activated at voltage positive to −40 mV, peak between −10 and 0 mV and inactivate with a slow decay when Ba 2+ ions (5 m m) are used as charges carrier. These properties correspond to those of the high voltage activated, DHP-sensitive, (L-type) Ca channel. No low voltage activated (T-type) currents have been evidenced. The present work also provides the first report about the modulation of Ca channels in adult human atrial cells by β-adrenergic agonists and dihydropyridines (agonists and antagonists). Electrophysiological and pharmacological properties of these Ca channels are qualitatively similar to those of the L-type Ca currents recorded from cardiac animal cells. However, at a physiological calcium concentration (2 m m), basal Ca currents are often very small or even absent but are revealed following the addition of the dihydropyridine (DHP) agonist Bay K 8644. Whether the decrease of the basal Ca current amplitude may be related to the chronic pretreatment of the patients by Ca channel blockers or to the pathology is discussed.

Characteristics of the time-dependent slow inward current in adult human atrial single myocytes

The slow inward current isi plays a prominent role in impulse formation and slow conduction in the human heart. For this reason, it may be involved in a great variety of arrhythmias that occur clinically, particularly at the supraventricular level. isi also appears to be the major target of numerous cardioactive drugs daily administered to many patients. Difficulties encountered in voltage clamping human tissues, inevitably dissected from restricted zones of myocardium with inadequate geometry, may explain why direct measurements of this current have never been performed on multicellular human preparations. The recent development of enzymatic techniques allowing the isolation of calcium-tolerant myocytes has provided a solution to these problems. To our knowledge only one abstract has presented until now records of isi in human ventricular myocytes. We describe here the result of experiments carried out to investigate the slow inward current in single cells isolated from adult human atria. The present work provides the first quantitative description of this current in human myocytes.

Fractionation and characterization of two molecular variants of myosin from adult human atrium

Monoclonal antibodies (MAb) to myosin heavy chains were prepared from one adult human ventricular myocardium. Several of these MAb reacted by indirect immunofluorescence in a heterogenous way on cryostat transverse sections of fibers from human atrial myocardium, suggesting the presence of different forms of myosin within the human atrium and prompting the further use of the MAb to attempt to fractionate preparations of native atrial myosins. Two molecular variants of human atrial myosins or myosin fragments were thus separated by immunoaffinity chromatography performed with one antiventricular myosin MAb. Seven MAb located at different positions along the myosin heavy chains, as deduced from blotting and immuno-electron microscopy experiments, were used to characterize the structural relationships between the separated human atrial isomyosins and between each of them and the main human ventricular myosin. As deduced from competitive radioimmunoassay measurements, the primary structures of the two atrial myosins differ in at least five antigenic determinants and share at least two of them; similarly located structural differences were observed between one of the atrial myosins and the ventricular myosin. Conversely, the primary structures of the other atrial myosin and of the ventricular myosin differ in at least two antigenic determinants and share at least five of them. Differences in the primary structures of the human cardiac myosins were confirmed by analysis of the peptides produced by limited enzymatic digestion of the heavy chains; a few peptide differences were consistently found. To summarize the two separated forms of atrial myosin have different heavy chains, but they have similar if not identical in vitro ATPase activities and the same light chains. One of the atrial myosins is immunologically close to the ventricular myosin, but they each differ with respect to their heavy chains, light chains, and enzymatic activities.

Foetal myosin light chain in human ventricle.

Myosin light chain subunits from adult human atria and ventricle were characterized by two-dimensional gel electrophoresis. Atrial (ALC-1 and ALC-2) differed from ventricular (VLC-1 and VLC-2) light chains. Foetal (18--21 week) ventricle contained VLC-1 plus a foetal light chain (FLC-1) which could not be distinguished from adult ALC-1. Myosin FLC-1 was gradually lost from the ventricle and replaced by VLC-1 during foetal development and in the immediate postnatal period.