Adult Atria Research Articles

Changes in cAMP signaling are associated with age-related downregulation of spontaneously beating atrial tissue energetic indices.

The prevalence of atria-related diseases increases exponentially with age and is associated with ATP supply-to-demand imbalances. Because evidence suggests that cAMP regulates ATP supply-to-demand, we explored aged-associated alterations in atrial ATP supply-to-demand balance and its correlation with cAMP levels. Right atrial tissues driven by spontaneous sinoatrial node impulses were isolated from aged (22-26months) and adult (3-4months) C57/BL6 mice. ATP demand increased by addition of isoproterenol or 3-Isobutyl-1-methylxanthine (IBMX) and decreased by application of carbachol. Each drug was administrated at the dose that led to a maximal change in beating rate (Xmax) and to 50% of that maximal change in adult tissue (X50). cAMP, NADH, NAD + NADH, and ATP levels were measured in the same tissue. The tight correlation between cAMP levels and the beating rate (i.e., the ATP demand) demonstrated in adult atria was altered in aged atria. cAMP levels were lower in aged compared to adult atrial tissue exposed to X50 of ISO or IBMX, but this difference narrowed at Xmax. Neither ATP nor NADH levels correlated with ATP demand in either adult or aged atria. Baseline NADH levels were lower in aged as compared to adult atria, but were restored by drug perturbations that increased cAMP levels. Reduction in Ca2+-activated adenylyl cyclase-induced decreased cAMP and prolongation of the spontaneous beat interval of adult atrial tissue to their baseline levels in aged tissue, brought energetics indices to baseline levels in aged tissue. Thus, cAMP regulates right atrial ATP supply-to-demand matching and can restore age-associated ATP supply-to-demand imbalance.

Changes in coexpression of pericytes and endogenous cardiac progenitor cells from heart development to disease state.

Focussing on the potential role of cardiovascular cell therapy, we investigated the spatial relationship between pericytes (cells with cardiac repair capabilities that ensheath blood vessels) and endogenous cardiac progenitors within stem cells' niches. We explored possible changes in their co-localisation in developing human hearts from foetal to adult stage and following ischaemia. Foetal and adult human heart specimens, obtained under ethical consent (University of Edinburgh ethics committee), were used for immunohistochemistry, cell isolation, culture and differentiation. Multi-lineage differentiation in culture, by single and double staining was completed for CD 146+ foetal pericytes and c-kit+ cells. Endothelial markers (CD31) gene expression was quantified by qPCR. c-kit+ cells frequency and coexpression with pericytes decrease with heart development, already evident by gestation week 19th. Pericytes and c-kit+ cells express the early cardiac transcription factors Nkx2.5 and Islet 1. Only c-kit+ cells express the stemness marker SSEA3 (24%), known to progressively decrease with cell differentiation. Endothelial differentiation assessment shows that cardiac pericytes and c-kit+ cells do not form CD31+ networks. This finding correlates with absence of staining for CD31 marker in both cultured cells' types. The cardiac marker α-actin was present in both cell populations. In healthy adult heart, pericyte markers CD146 localise within the vasculature. Following ischaemia this pericyte marker becomes also evident outside the vasculature.In healthy adult atrium, c-kit expression is low and coexpression with other markers inconspicuous. Ischaemia leads to increased c-kit expression, particularly in blood vessels <50um diameter. Furthermore, following ischaemia c-kit, endothelium and pericyte markers co-localise within the same atrial cells. Blood vessels >50μm diameter showed mostly only staining for endothelial (vWF) and pericyte (CD146) markers, with no co-expression of c-kit marker identified. Staining patterns within the ischaemic regions of the right and left atrial appendages revealed low levels of colocalisation between vWF and CD146. Acute ischaemia of the left ventricle affected the detection of cardiac stem cells markers in the area of injury, due to myocardium disruption. Foetal heart pericytes and c-kit+ cells express early cardiac transcription factors and show trans-differentiation potential, which decreases in healthy adult hearts. The preservation and activity of cardiac stem cells niches within the atrium vasculature appears re-activated in post-ischaemic hearts. Better understanding of cardiac c-kit+ and pericyte cells during-human embryonic development and during ischaemia may identify alternative novel therapeutic strategy against coronary artery disease.

Atrial natriuretic peptide regulates adipose tissue accumulation in adult atria

The abundance of epicardial adipose tissue (EAT) is associated with atrial fibrillation (AF), the most frequent cardiac arrhythmia. However, both the origin and the factors involved in EAT expansion are unknown. Here, we found that adult human atrial epicardial cells were highly adipogenic through an epithelial-mesenchymal transition both in vitro and in vivo. In a genetic lineage tracing the WT1CreERT2+/-RosatdT+/- mouse model subjected to a high-fat diet, adipocytes of atrial EAT derived from a subset of epicardial progenitors. Atrial myocardium secretome induces the adipogenic differentiation of adult mesenchymal epicardium-derived cells by modulating the balance between mesenchymal Wingless-type Mouse Mammary Tumor Virus integration site family, member 10B (Wnt10b)/β-catenin and adipogenic ERK/MAPK signaling pathways. The adipogenic property of the atrial secretome was enhanced in AF patients. The atrial natriuretic peptide secreted by atrial myocytes is a major adipogenic factor operating at a low concentration by binding to its natriuretic peptide receptor A (NPRA) receptor and, in turn, by activating a cGMP-dependent pathway. Hence, our data indicate cross-talk between EAT expansion and mechanical function of the atrial myocardium.

Endothelial–mesenchymal transition in human atrial fibrillation

BackgroundAtrial fibrosis is a hallmark of atrial structural remodeling leading to the persistence of atrial fibrillation. Although fibroblasts play a major role in atrial fibrosis, their source in the adult atrium is unclear. We tested the hypothesis that endothelial cells contribute to fibroblast accumulation through an endothelial–mesenchymal transition in the atrium of patients with atrial fibrillation. Methods and resultsThe study group consisted of patients with atrial fibrillation and valvular disease or atrial septal defect who underwent left atrial appendectomy during cardiac surgery (n=38). The amount of fibrotic depositions in the left atrium positively correlated with left atrial dimension. Furthermore, snail and S100A4, indicative of endothelial–mesenchymal transition, were quantified in the left atrium using western blot analysis, which showed statistically significant correlations with left atrial dimension. Immunofluorescence assay of the left atrial tissue identified snail and S100A4 being expressed within the endocardium which is composed of CD31+ cells. The snail-positive endocardium also showed the expression of membrane type 1-matrix metalloproteinase. Immunofluorescence multi-labeling experiments identified that heat shock protein 47, prolyl-4-hydroxylase, and procollagen type 1 co-localized with snail and S100A4 within the endothelial cells of the left atrium, indicating the mesenchymal phenotype to produce collagen. ConclusionsIn this study, we showed that the endothelial–mesenchymal transition occurs in the atrium of patients with atrial fibrillation. This observation should help in constructing a novel therapeutic approach for preventing atrial structural remodeling.

Abstract 16157: A TBX5 Driven Gene Regulatory Network Prevents Atrial Fibrillation

Atrial fibrillation (AF) is the most common cardiac arrhythmia. The genetics of AF are complex, but recent GWAS have revealed a number of AF risk loci, including TBX5 . TBX5 plays a critical role in heart development, and continues to be expressed in the adult atria. We assessed the role of Tbx5 in the adult heart by removing Tbx5 from the mouse at 6 weeks-of-age. Adult Tbx5 mutant mice developed spontaneous sustained atrial fibrillation, characterized by surface electrocardiogram (ECG), intracardiac ECGs, and ex vivo whole heart optical mapping. Tbx5 mutant isolated cardiomycytes showed prolonged action potentials, disrupted calcium handling, and abnormal automaticity. Tbx5 mutant atrial gene expression analysis revealed down-regulation of numerous conduction system effectors genes including Scn5a, Ryr2, Gja1, Atp2a2 , Dsp , and numerous potassium channels. We also observed Tbx5 dependence of the transcription factor gene Pitx2 , the number one locus associated with AF by GWAS. We identified an enhancer upstream of PITX2 that interacts with the PITX2 promoter by 4C and whose activity is TBX5 dependent and modulated by common SNP rs1906595, which alters a canonical TBX5 binding site. We also identified cis-regulatory elements at Scn5a, Ryr2, Gja1, Atp2a2 , and Dsp , locus co-regulated by Tbx5 and Pitx2. We describe a TBX5-driven atrial molecular network including Pitx2 that determines atrial conduction properties.

YIA3 Regional Action Potential Gradients in the Murine Left Atrium

Background Regional differences in action potential (AP) morphology in the adult left atrium (LA) may account for susceptibility to atrial fibrillation. The posterior LA wall close to the junction with the pulmonary veins (LA-PV) is an important area contributing to initiation and maintenance of atrial fibrillation. The posterior LA has a distinct embryologic origin: Its development is regulated by PITX2, a gene that has been inflicted in the genetic predisposition to AF. So far, it is not known whether the electrophysiology of the posterior LA differs from that of other parts of the LA. We therefore characterised the electrical phenotype of the LA-PV compared to other LA regions. Methods Transmembrane (TAPs) and optical (OAPs) action potentials were recorded from 20 superfused murine LA taken from 12 male and 8 female mice aged 12–20 W, bred on an MF1 background. Optical high-density AP mapping was performed and optical APs were recorded from 9 regions across the LA using a dedicated murine activation system (Hamamatsu ORCA flash 4, Di-4-Anepps, sampling rate 1–2 kHz). OAPs were analysed using custom made algorithms produced in MATLAB. At each cycle length, 25 action potentials were averaged to generate a mean waveform that was then used for analysis. TAPs were recorded from 3 LA regions: 1) the posterior LA towards the junction with the pulmonary vein (LA-PV), 2) the medial dome (LA-M) and 3) the LA lateral wall (LA-LW). All Preparations were paced at 100ms. Results OAP distribution maps (n = 8 LA) identified a LA-PV to LA-LW pattern of APD shortening and also demonstrated that there is relatively high local APD heterogeneity within the LA-PV region. TAP recordings identified a higher action potential amplitude (APA) in the LA-PV (84 ± 2 mV) than LA-M (78 ± 1 mV, p Conclusion Regional AP differences in the murine LA clearly suggest that the posterior left atrium has a distinct electrophysiological profile which may render this region susceptible to the genesis and maintenance of AF. Prolonged APD and higher AP amplitude suggest a role as a driver of electrical LA activity. Further studies are warranted to determine whether PITX2 is important for the maintenance of these electrophysiological differences.

Abstract 18782: Regional Action Potential Heterogeneity in the Murine Pitx2c Deficient Left Atrium

Introduction and hypothesis: Genetic abnormalities close to the Pitx2 gene correlate more strongly with atrial fibrillation (AF) than any other genetic modification. It is known that Pitx2c mRNA is expressed in the adult left atrium (LA), but its relevance for the electrical integrity of cardiomyocytes throughout the atria remains unresolved. We therefore compared regional action potential (AP) morphology in PITX2c +/- and wild type (WT) littermate mice. Methods: Transmembrane (T) and optical (O) APs were recorded from superfused intact, isolated LA paced at 100ms. TAPs were recorded from three distinct regions on the LA epicardium: 1) the junction with the interatrial septum (S), 2) the medial muscular dome (M) and 3) the lateral wall (L). OAPs were recorded using a custom murine atrial optical mapping system using the Hamamatsu ORCA flash 4 and Di-4-Anepps. Results: OAPs were shorter in Pitx2c deficient LA (AP duration (APD70); WT 13±2ms, n=4 LA vs Pitx2c +/- 9±1ms, n=4 LA, all data as mean±SEM, p&lt;0.05). Simultaneous recordings of OAPs from multiple LA regions showed an AP gradient with longer OAPs at the septum than at the lateral wall, in both genotypes. In WT LA, TAPs were longer and of greater amplitude at the septum than in the other two regions (APD90; S 26±1ms, M 19±1mV and L 19±1ms, APA; S 81±1mV, M 76±2mV and L 76±2mV, n=6 LA, p&lt;0.05). In Pitx2c +/- , TAPs were shorter than WT in all three regions tested and exhibited the same regional patterning (S 20±1ms, M 15±1ms and L 16±1, n=7 LA, p&lt;0.05). The regional AP amplitude gradient was not apparent, or at least shifted more towards the lateral wall, in PITX2c +/- atria (APA; S 77±2mV, M 76±3mV and L 74±2mV, n=7 LA, ns). Conclusion: Partial depletion in Pitx2c mRNA expression causes a uniform APD shortening throughout the LA and loss of regional changes in AP amplitude. Identification of the targets downstream of Pitx2 accounting for these changes will help to better understand the mechanisms conveying AF due to reduced Pitx2 function.

244 EXPLORING PUTATIVE PITX2C-DEPENDENT GENES IN MOUSE ATRIA

Paired-like homeodomain transcription factor 2 (<i>Pitx2</i>) is involved in the embryonic left-right (L-R) patterning of heart and lung. So far, there is no known function for PITX2 in the adult heart. We recently demonstrated that <i>Pitx2c</i> is expressed highly and selectively in left atria of adult mice and humans compared to the right atria (Kirchhof <i>et al</i>., 2011), indicative of a functional role for <i>Pitx2c</i> in the adult left atrium. Furthermore, there is a strong association between polymorphisms close to the <i>PITX2</i> locus (on chromosome 4q25) and atrial fibrillation (AF) (Gudbjartsson <i>et al</i>., 2007; Kaab <i>et al</i>., 2009). We hypothesise that alterations in the L-R atrial expression ratio could promote the development of ectopic stimuli and AF. Gene array analysis previously identified a number of genes differentially regulated between left and right atria in wildtype adult mice (Kahr <i>et al</i>., 2011). Indirect comparison with gene arrays performed in conjunction with these experiments, suggested that <i>Ccl21, Ddit4l and Ppp1r1b</i> were downregulated in the left atria of <i>Pitx2c</i> heterozygous mice, suggestive of downstream Pitx2-dependent expression in the adult left atrium. In this current work we have sought to quantify differential expression levels of Chemokine ligand 21 (<i>Ccl21</i>); DNA damage-inducible transcript 4-like (<i>Ddit4l</i>); Pleckstrin homology-like domain family A, member 1 (<i>Phlda1</i>); Protein phosphatase 1 regulatory subunit 1B (<i>Ppp1r1b</i>); Scavenger receptor class A (<i>Scara5</i>); Troponin I2 type 2 (<i>Tnni2</i>) and Chemokine ligand 14 (<i>Cxcl14</i>) by RT-PCR in the left and right atria of 18 mice (9 <i>Pitx2c</i> heterozygous and 9 litter mate controls) aged between 14-20wks. RT-PCR was performed using SYBR Green and the ΔΔCt Method. All genes were found to be differentially expressed between the left and right atria in both wildtype and <i>Pitx2c</i> heterozygous mice. There was a trend towards differential fold change reduction in the left atria of <i>Pitx2c</i> heterozygous mice compared to wildtype for all candidate genes. The L-R ratios between <i>Ccl21</i> and <i>Ddit4l</i> differ significantly (<i>Ccl21</i> 0.372, p=0.01; <i>Ddit4l</i> 0.275, p=0.04). <i>Ddit4l</i> encodes a protein involved in DNA-damage and hypoxia-induced cell death whilst disruption of the CCL21 pathway in mice improved myocardial dysfunction suggesting that CCL21 levels actively impact upon cardiac pathology. These results support the hypothesis that <i>Ccl21</i> and <i>Ddit4l</i> are positively regulated by <i>Pitx2c</i>, and may suggest that <i>Pitx2c</i> maintains leftness in the adult atrium; hence the contribution of these genes to AF pathology warrants further investigation.

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

Purification, Expansion and Characterization of Putative Murine Cardiac Progenitor Cells

Ischemic heart disease kills more people than any other condition. Medical treatment for ischemic heart disease is currently limited by the heart’s lack of regeneration after injury. Recent reports have suggested the existence of cardiac progenitor cells in the adult normal and diseased mammalian heart. The origin of these cells is unclear. We implemented novel culturing conditions to isolate putative cardiac progenitor cells (pCPCs) from the adult murine heart atrium with the properties of cardiac regeneration. Putative adult cardiac progenitor cells were purified and expanded from adult murine hearts by expansion medium supplemented with 2% fetal calf serum, epidermal growth factor, platelet-derived growth factor and leukemia inhibitory factor. Under the culture condition of 5% O2 , these cells can be expanded beyond 42 days, expressed stem cell antigen (Sca-1) and cardiac-specific transcription factors. When treated with oxytocin in vitro, these cells express cardiac contractile proteins and when injected intramuscularly in the tibialis anterior muscle these cells give rise to cardiomyocyte-like cells. In contrast, transplantation of these pCPCs in uninjured heart did not result in cardiomyocyte differentiation suggesting that the heart environment is less permissive than the skeletal muscle of cardiogenesis. These results suggest that cells from the adult murine heart selected with this culture conditions and transplanted in the skeletal muscle have cardiogenic potential. Thus, this approach warrants further investigation for the therapeutic development of strategies for cardiac tissue engineering and myocardial regeneration.

Difference of Sodium Currents between Pediatric and Adult Human Atrial Myocytes: Evidence for Developmental Changes of Sodium Channels

Voltage-gated calcium currents and potassium currents were shown to undergo developmental changes in postnatal human and animal cardiomocytes. However, so far, there is no evidence whether sodium currents also presented the developmental changes in postnatal human atrial cells. The aim of this study was to observe age-related changes of sodium currents between pediatric and adult atrial myocytes. Human atrial myocytes were acutely isolated and the whole-cell patch clamp technique was used to record sodium currents isolated from pediatric and adult atrial cardiomocytes. The peak amplitude of sodium currents recorded in adult atrial cells was significantly larger than that in pediatric atrial myocytes. However, there was no significant difference of the activation voltage for peak sodium currents between two kinds of atrial myocytes. The time constants for the activation and inactivation of sodium currents were smaller in adult atria than pediatric atria. The further study revealed that the voltage-dependent inactivation of sodium currents were more slow in adult atrial cardiomyocytes than pediatric atrial cells. A significant difference was also observed in the recovery process of sodium channel from inactivation. In summary, a few significant differences were demonstrated in sodium currents characteristics between pediatric and adult atrial myocytes, which indicates that sodium currents in human atria also undergo developmental changes.

Adult zebrafish heart as a model for human heart? An electrophysiological study

The zebrafish has recently emerged as an excellent model for studies of heart development and regeneration. The physiology of the zebrafish heart has been suggested to resemble that of the human heart in many aspects, whereas, in contrast to mammals, the zebrafish has a remarkable ability to regenerate after heart injury. Thus, zebrafish have been proposed as a cost-effective model for genetic and pharmacological screens of factors affecting heart function and repair. However, realizing the full potential of the zebrafish heart as a model will require a better understanding of the electrophysiology of the adult zebrafish myocardium. Here, we characterize action potentials (APs) from intact adult atria and ventricles and find that the overall shape of zebrafish APs is similar to that of humans. We show that zebrafish, like most mammals, display functional acetylcholine-activated K + channels in the atrium, but not in the ventricle. Furthermore, the zebrafish AP upstroke is dominated by Na + channels, L-type Ca 2+ channels contribute to the plateau phase and I Kr channels are involved in repolarization. However, despite these similarities between zebrafish and mammalian electrophysiology, we also identified important differences. In particular, zebrafish display a robust T-type Ca 2+ current in both atrial and ventricular cardiomyocytes. Interestingly, in most mammals T-type Ca 2+ channels are only expressed in the developing heart or under pathophysiological conditions, indicating that adult zebrafish cardiomyocytes display a more immature phenotype.

Development of Newer Calcium Channel Antagonists

Calcium channel antagonists are most frequently prescribed for the treatment of hypertension and the majority specifically inhibit the L-type Ca2+ channel. In order to prevent reflex sympathetic over activity caused by L-type calcium channel antagonists (calcium channel blockers [CCBs]), increasing attention has focused on the blockade of the T-type Ca2+ channel. The T-type Ca2+ channel is found in the kidney and can also appear in the ventricle of the heart when in failure. Therefore, the T-type Ca2+ channel is a possible new target for the treatment of nephropathy and heart failure. In clinical trials, the efficacy and safety of T-type CCBs in hypertension and chronic renal disease have been reported. It is well known that the T-type Ca2+ channel is present in the adult atrium and plays a role in the cardiac pacemaker, but recent experimental studies suggest that this current also promotes electrical remodelling of the atrium. Using efonidipine, a dual L- and T-type CCB, it has been demonstrated that atrial electrical remodelling can be diminished in dogs. Furthermore, the T-type Ca2+ channel has recently been found in the pulmonary veins, contributing to the pulmonary vein pacemaker activity and triggered activity. A variety of drugs having T-type CCB effects have been shown to be effective in the management of atrial fibrillation, suggesting that this channel may be a novel therapeutic target.

Sodium current function in adult and aged canine atrial cells

The incidence of atrial fibrillation increases with age, but it is unknown whether there are changes in the intrinsic function of Na+ currents in cells of the aged atria. Thus, we studied right (RA) and left (LA) atrial cells from two groups of dogs, adult and aged (>8 yr), to determine the change in Na+ currents with age. In this study all dogs were in normal sinus rhythm. Whole cell voltage clamp techniques were used to compare the Na+ currents in the two cell groups. Immunocytochemical studies were completed for the Na+ channel protein Na(v)1.5 to determine whether there was structural remodeling of this protein with age. In cells from aged animals, we found that Na+ currents are similar to those we measured in adult atria. However, Na+ current (I(Na)) density of the aged atria differed depending on the atrial chamber with LA cell currents being larger than RA cell currents. Thus with age, the difference in I(Na) density between atrial chambers remains. I(Na) kinetic differences between aged and adult cells included a significant acceleration into the inactivated state and an enhanced use-dependent decrease in peak current in aged RA cells. Finally, there is no structural remodeling of the cardiac Na+ channel protein Na(v)1.5 in the aged atrial cell. In conclusion, with age there is no change in I(Na) density, but there are subtle kinetic differences contributing to slight enhancement of use dependence. There is no structural remodeling of the fast Na+ current protein with age.

Differential Sialylation Modulates Voltage-gated Na+ Channel Gating throughout the Developing Myocardium

Voltage-gated sodium channel function from neonatal and adult rat cardiomyocytes was measured and compared. Channels from neonatal ventricles required an ∼10 mV greater depolarization for voltage-dependent gating events than did channels from neonatal atria and adult atria and ventricles. We questioned whether such gating shifts were due to developmental and/or chamber-dependent changes in channel-associated functional sialic acids. Thus, all gating characteristics for channels from neonatal atria and adult atria and ventricles shifted significantly to more depolarized potentials after removal of surface sialic acids. Desialylation of channels from neonatal ventricles did not affect channel gating. After removal of the complete surface N-glycosylation structures, gating of channels from neonatal atria and adult atria and ventricles shifted to depolarized potentials nearly identical to those measured for channels from neonatal ventricles. Gating of channels from neonatal ventricles were unaffected by such deglycosylation. Immunoblot gel shift analyses indicated that voltage-gated sodium channel α subunits from neonatal atria and adult atria and ventricles are more heavily sialylated than α subunits from neonatal ventricles. The data are consistent with approximately 15 more sialic acid residues attached to each α subunit from neonatal atria and adult atria and ventricles. The data indicate that differential sialylation of myocyte voltage-gated sodium channel α subunits is responsible for much of the developmental and chamber-specific remodeling of channel gating observed here. Further, cardiac excitability is likely impacted by these sialic acid–dependent gating effects, such as modulation of the rate of recovery from inactivation. A novel mechanism is described by which cardiac voltage-gated sodium channel gating and subsequently cardiac rhythms are modulated by changes in channel-associated sialic acids.

HKChIP2 is a functional modifier of hKv4.3 potassium channels: cloning and expression of a short hKChIP2 splice variant.

The Ca(2+) independent transient outward K(+) current (I(to1)) in the heart is responsible for the initial phase of repolarization. The hKv4.3 K(+) channel alpha-subunit contributes to the I(to1) current in many regions of the human heart. Consistently, downregulation of hKv4.3 transcripts in heart failure and atrial fibrillation is linked to reduction in I(to1) conductance. The recently cloned KChIP family of calcium sensors has been shown to modulate A-type potassium channels of the Kv4 K(+) channel subfamily. We describe the cloning and tissue distribution of hKChIP2, as well as its functional interaction with hKv4.3 after expression in Xenopus oocytes. Furthermore, we isolated a short splice variant of the hKChIP2 gene (hKCNIP2), which represents the major hKChIP2 transcript. Northern blot analyses revealed that hKChIP2 is expressed in the human heart and occurs in the adult atria and ventricles but not in the fetal heart. Upon coexpression with hKv4.3 both hKChIP2 isoforms increased the current amplitude, slowed the inactivation and increased the recovery from inactivation of hKv4.3 currents. For the first time we analyzed the influence of a KChIP protein on the voltage of half-maximal inactivation of Kv4 channels. We demonstrate that the hKChIP2 isoforms shifted the half-maximal inactivation to more positive potentials, but to a different extent. By elucidating the genomic structure, we provide important information for future analysis of the hKCNIP2 gene in candidate disorders. In the course of this work we mapped the hKCNIP2 gene to chromosome 10q24. Heteromeric hKv4.3/hKChIP2 currents more closely resemble native epicardial I(to1), suggesting that hKChIP2 is a true beta-subunit of human cardiac I(to1). As a result hKChIP2 might play a role in cardiac diseases, where a contribution of I(to1) has been shown.

The physiological and pathophysiological modulation of the endocrine function of the heart

Under physiological conditions, the endocrine heart contributes to the maintenance of cardiovascular homeostasis through the polypeptide hormones ANF and BNP, which are members of the natriuretic peptide (NP) family. Given that NPs are of interest from the basic and clinical points of view, the genetic expression and secretion of ANF and BNP as well as the nature of the interaction of these hormones with their receptors has been the subject of extensive studies since the discovery of ANF in 1980. Following hemodynamic overload, increased secretion of NPs by the heart can be seen. This change may occur without an increase in gene expression as observed for atrial NPs following acute volume expansion, or it can occur with an increase in both ANF and BNP gene expression in atria only as seen in mineralocorticoid escape during which it is obvious that a critical decrease in hormone stores must be reached before transcriptional activation occurs. Chronic hemodynamic pressure or volume overload results in increased expression of NPs in atria and ventricles. Under these circumstances, the increased production of BNP by hypertrophic ventricles changes the normal plasma concentration ratio of ANF to BNP, a fact that has clinical diagnostic and prognostic implications. There are exceptions to this rule: chronic, severe L-NAME hypertension, which may occur without left ventricular hypertrophy, does not cause this effect and increased ventricular NP gene expression can occur in mineralocorticoid hypertension before detectable ventricular hypertrophy. Atrial and ventricular NP gene expression appears to be under different transcriptional control because pharmacological treatments such as chronic ACE inhibition or ET(A) receptor blockade can reverse the increased ventricular NP expression but has no detectable effect on atrial NP gene expression. This is not unlike the myosin heavy chain switch that is observed in certain pathologies, and can be pharmacologically reversed in a manner similar to NPs in the ventricles but it does not occur in atrial muscle. These observations made in vivo or using isolated adult atria often differ strikingly from results obtained using the mixed phenotype afforded by cardiocytes in culture, indicating that the kinds of questions addressed by each approach must be judiciously chosen. G-protein coupled receptor-mediated actions of neurohumors such as endothelin and phenylephrine are normally used to stimulate NP gene expression and release in different in vitro models. The main physiological stimulus for increased ANF release, atrial muscle stretch, also appears to rely on G-protein-coupled mechanisms. Alternative agonists and receptor types at play are suggested by the finding that circulating levels of BNP are selectively increased before and during overt cardiac allograft rejection episodes in human patients. The data suggest that enhanced BNP plasma levels could form a basis for a noninvasive test for cardiac allograft rejection. However, the molecular mechanism by which expression of NPs are regulated in the transplanted heart is not well understood. Conditioned medium from mixed lymphocyte reaction cultures, considered an in vitro model of transplantation immunity, induces specific upregulation of BNP as do individual pro-inflammatory cytokines. Findings such as these suggest that the study of NPs will continue to produce a wealth of information relevant to basic and clinical scientists.

Human cardiac myosin heavy chain isoforms in fetal and failing adult atria and ventricles.

The goal of this study was to test the hypothesis that the relative amounts of the cardiac myosin heavy chain (MHC) isoforms MHC-alpha and MHC-beta change during development and transition to heart failure in the human myocardium. The relative amounts of MHC-alpha and MHC-beta in ventricular and atrial samples from fetal (gestational days 47--110) and nonfailing and failing adult hearts were determined. The majority of the fetal right and left ventricular samples contained small relative amounts of MHC-alpha (mean < 5% of total MHC). There was a small significant decrease in the level of MHC-alpha in the ventricles between 7 and 12 wk of gestation. Fetal atria expressed predominantly MHC-alpha (mean > 95%), with MHC-beta being detected in most samples. The majority of adult nonfailing right and left ventricular samples had detectable levels of MHC-alpha ranging from 1 to 10%. Failing right and left ventricles expressed a significantly lower level of MHC-alpha. MHC-alpha comprised approximately 90% of the total MHC in adult nonfailing left atria, whereas the relative amount of MHC-alpha in the left atria of individuals with dilated or ischemic cardiomyopathy was approximately 50%. The differences in MHC isoform composition between fetal and nonfailing adult atria and between fetal and nonfailing adult ventricles were not statistically significant. We concluded that the MHC isoform compositions of fetal human atria are the same as those of nonfailing adult atria and that the ventricular MHC isoform composition is different between adult nonfailing and failing hearts. Furthermore, the marked alteration in atrial MHC isoform composition, associated with cardiomyopathy, does not represent a regression to a pattern that is uniquely characteristic of the fetal stage.

The physiological and pathophysiological modulation of the endocrine function of the heart

Under physiological conditions, the endocrine heart contributes to the maintenance of cardiovascular homeostasis through the polypeptide hormones ANF and BNP, which are members of the natriuretic peptide (NP) family. Given that NPs are of interest from the basic and clinical points of view, the genetic expression and secretion of ANF and BNP as well as the nature of the interaction of these hormones with their receptors has been the subject of extensive studies since the discovery of ANF in 1980. Following hemodynamic overload, increased secretion of NPs by the heart can be seen. This change may occur without an increase in gene expression as observed for atrial NPs following acute volume expansion, or it can occur with an increase in both ANF and BNP gene expression in atria only as seen in mineralocorticoid escape during which it is obvious that a critical decrease in hormone stores must be reached before transcriptional activation occurs. Chronic hemodynamic pressure or volume overload results in increased expression of NPs in atria and ventricles. Under these circumstances, the increased production of BNP by hypertrophic ventricles changes the normal plasma concentration ratio of ANF to BNP, a fact that has clinical diagnostic and prognostic implications. There are exceptions to this rule: chronic, severe L-NAME hypertension, which may occur without left ventricular hypertrophy, does not cause this effect and increased ventricular NP gene expression can occur in mineralocorticoid hypertension before detectable ventricular hypertrophy. Atrial and ventricular NP gene expression appears to be under different transcriptional control because pharmacological treatments such as chronic ACE inhibition or ETA receptor blockade can reverse the increased ventricular NP expression but has no detectable effect on atrial NP gene expression. This is not unlike the myosin heavy chain switch that is observed in certain pathologies, and can be pharmacologically reversed in a manner similar to NPs in the ventricles but it does not occur in atrial muscle. These observations made in vivo or using isolated adult atria often differ strikingly from results obtained using the mixed phenotype afforded by cardiocytes in culture, indicating that the kinds of questions addressed by each approach must be judiciously chosen. G-protein coupled receptor-mediated actions of neurohumors such as endothelin and phenylephrine are normally used to stimulate NP gene expression and release in different in vitro models. The main physiological stimulus for increased ANF release, atrial muscle stretch, also appears to rely on G-protein-coupled mechanisms. Alternative agonists and receptor types at play are suggested by the finding that circulating levels of BNP are selectively increased before and during overt cardiac allograft rejection episodes in human patients. The data suggest that enhanced BNP plasma levels could form a basis for a noninvasive test for cardiac allograft rejection. However, the molecular mechanism by which expression of NPs are regulated in the transplanted heart is not well understood. Conditioned medium from mixed lymphocyte reaction cultures, considered an in vitro model of transplantation immunity, induces specific upregulation of BNP as do individual pro-inflammatory cytokines. Findings such as these suggest that the study of NPs will continue to produce a wealth of information relevant to basic and clinical scientists.Key words: atrial natriuretic factor, hypertension, hypertrophy, heart failure, cytokines.

Complete sequence and characterization of chick ventricular myosin heavy chain in the developing atria

We isolated five complementary DNA (cDNA) clones, encoding the chick ventricular myosin heavy chain (MyHC) by reverse transcription polymerase chain reaction (RT-PCR). The entire cDNA consists of 5995 nucleotides with the 52 bp 5′-untranslated region and the 129 bp 3′-untranslated region. The complete cDNA encodes 1937 amino acids. Expression of the chick ventricular MyHC gene was also studied by Northern blot analysis. This gene continued to be strongly expressed in the ventricle during cardiac development. On the other hand, its expression was moderate in the early embryonic atria, and was down-regulated during development. In the adult atria, this gene was expressed at very low levels. To determine the localization of the ventricular MyHC protein, an immunohistochemical study was performed. The ventricular MyHC was present in early embryonic atrial myocytes. During development, the expression of this protein in the atrial myocytes was down-regulated, but continued to be present in the atrial conduction system. Our results indicate that the ventricular MyHC appears in the primary atrial myocardium and is then localized in the conduction cells of the atria.