Membrane-associated peptidylglycine alpha-amidating monooxygenase in the heart

Recent investigations have shown that the heart atrium is an endocrine tissue. In the present studies, high levels of peptidylglycine alpha-amidating monooxygenase (PAM), which catalyzes the formation of bioactive alpha-amidated peptides from their glycine-extended precursors, have been found in particulate fractions from bovine and rat heart atrium; only low levels of PAM activity were present in soluble fractions. Corresponding fractions from the ventricles contained 20-fold less activity. Immunocytochemical studies demonstrated that PAM was localized primarily to atrial cardiocytes, with a distribution resembling that of atriopeptin. Following differential centrifugation of rat atrial homogenates, most of the PAM activity was associated with crude granule fractions, with lesser amounts of activity associated with crude microsomal fractions. Upon further subcellular fractionation, PAM activity in the rat atrium was found primarily with immunoactive atriopeptin in fractions enriched in secretory granules. Following sodium dodecyl sulfate-polyacrylamide gel electrophoresis, antisera to purified bovine pituitary PAM identified a 113,000-dalton protein in bovine atrial microsomes and secretory granules; the protein predicted from the sequence of the cDNA encoding bovine pituitary PAM is of similar size (Eipper, B. A., Park, L. P., Dickerson, I. M., Keutmann, H. T., Thiele, E. A., Rodriguez, H., Schofield, P. R., and Mains, R. E. (1987) Mol. Endocrinol. 1, 777-790). Northern blot analysis using cDNA probes encoding bovine pituitary PAM demonstrated higher levels of PAM mRNA in heart atrium than in anterior pituitary. Rat heart contains PAM mRNA species of 3.6 and 3.8 kilobases, the smaller mRNA species corresponding in size to the PAM mRNA expressed in rat anterior pituitary.     

Chromogranin A: Immunocytochemical localization in secretory granules of frog atrial cardiomyocytes

Chromogranin A (CgA) belongs to the granin family of acidic proteins that are present in the secretory granules of many endocrine, neuroendocrine, and nerve cells. CgA has been shown to be stored in cardiomyocyte secretory granules of the rat heart atrium together with atrial natriuretic peptide (ANP). CgA-derived peptides (vasostatins) are known to produce a cardiosuppressive effect on isolated and working in vitro frog and rat hearts. Recently, CgA-derived vasostatin-containing peptides have been identified in rat hearts, whereas no data are available so far about the presence of CgA in the frog heart. In our work, we have studied the subcellular CgA localization in atrial myocytes of the adult frog R. temporaria heart by using an ultraimmunocytochemical method. Immunocytochemical staining of the frog atrial tissue for CgA and ANP showed the presence of the CgA-immunoreactive material in two types (A and B) of large specific atrial secretory granules, whereas no gold particles were revealed over the small granules (D) with a high electron density core. Similar results were obtained during the immunocytochemical staining by an antibody to ANP of the drog atrial cardiomyocytes. The data of the present work allow for the suggestion that CgA revealed in frog atrial cardiomyocytes, like CgA in rat cardiomyocytes, can be considered to be a precursor of intracardial vasostatins that, together with ANP, can play an important cardioprotector role under conditions of stress.     

Differentiation of endocrine myocardiocytes in the developing heart of the toad (Bufo arenarum Hensel).

The differentiation of endocrine myocardiocytes was investigated in the heart of developing toad Bufo arenarum Hensel, combining ultrastructural and immunocytochemical procedures. The distribution of immuno-reactive atrial natriuretic peptide (ANP) in the whole heart was appraised by light microscopy, applying biotin-streptavidin and immunofluorescence techniques. With the latter procedures ANP was first recognized at embryonic stage 22, in both atrium and ventricle. In the ensuing stages the ANP-reactivity became stronger in the atrium, while it became dimmer in the ventricle. At the end of the larval prometamorphic stage, atrial myocardiocytes acquired almost all the features of adult myoendocrine cells. At electron microscope level, small inclusions, about 110-120 nm in diameter, resembling secretory granules were found in myoendocrine cells beginning at embryonic stage 22. However, no immunogold labeling of ANP occurred until stage 25. The number of secretory granules diminished in the ventricles and increased in the atrium of the larval heart and at the end of the prometamorphic stage the atrial myoendocrine cells presented the ultrastructural characteristics of active secretory cells. The synthesis of ANP in larvae is enhanced at a critical period of development when the developing toad switches from an aquatic environment to terrestrial life. The cardiac hormones seem to play a key role in the regulation of the osmolarity of body fluids at this developmental stage.     

Cloning and expression of a new inositol 1,4,5-trisphosphate receptor type 1 splice variant in adult rat atrial myocytes

Inositol 1,4,5-trisphosphate receptor type 1 (IP₃R1) is already known to be highly expressed in the brain, and is found in many other tissues, including the atrium of the heart. Although the complete primary structure of IP₃R1 in the rat brain has been reported, the complete sequence of an IP₃R1 clone from atrial myocytes has not been reported. We isolated an IP₃R1 complementary DNA (cDNA) clone from isolated adult rat atrial myocytes, and found a new splice variant of IP₃R1 that was different from a previously reported IP₃R1 cDNA clone obtained from a rat brain (NCBI GenBank accession number: NM_001007235). Our clone had 99% similarity with the rat brain IP₃R1 sequence; the exceptions were 39 amino acid deletions at the position of 1693–1731, and the deletion of phenylalanine at position 1372 that lay in the regulatory region. Compared with the rat brain IP₃R1, in our clone proline was replaced with serine at residue 2439, and alanine was substituted for valine at residue 2445. These changes lie adjacent to or within the fifth transmembrane domain (2440–2462). Although such changes in the amino acid sequences were different from the rat brain IP3R1 clone, they were conserved in human or mouse IP3R1. We produced a plasmid construct expressing the atrial IP3R1 together with green fluorescent protein (GFP), and successfully overexpressed the atrial IP3R1 in the adult atrial cell line HL-1. Further investigation is needed on the physiological significance of the new splice variant in atrial cell function.     

Rat corin gene: molecular cloning and reduced expression in experimental heart failure

Stored cardiac pro-atrial natriuretic peptide (pro-ANP) is converted to ANP and released upon stretch from the atria into the circulation. Corin is a serin protease with pro-ANP-converting properties and may be the rate-limiting enzyme in ANP release. This study was aimed to clone and sequence corin in the rat and to analyze corin mRNA expression in heart failure when ANP release upon stretch is blunted. Full-length cDNA of rat corin was obtained from atrial RNA by RT-PCR and sequenced. Tissue distribution as well as regulation of corin mRNA expression in the atria were determined by RT-PCR and RNase protection assay. Heart failure was induced by an infrarenal aortocaval shunt. Stretch was applied to the left atrium in a working heart modus, and ANP was measured in the perfusates. The sequence of rat corin cDNA was found to be 93.6% homologous to mouse corin cDNA. Corin mRNA was expressed almost exclusively in the heart with highest concentrations in both atria. The aortocaval shunt led to cardiac hypertrophy and heart failure. Stretch-induced ANP release was blunted in shunt animals (control 1,195 +/- 197 fmol.min(-1).g(-1); shunt: 639 +/- 99 fmol.min(-1).g(-1), P < 0.05). Corin mRNA expression was decreased in both atria in shunt animals [right atrium: control 0.638 +/- 0.004 arbitrary units (AU), shunt 0.566 +/- 0.014 AU, P < 0.001; left atrium: control 0.564 +/- 0.009 AU, shunt 0.464 +/- 0.009 AU, P < 0.001]. Downregulation of atrial corin mRNA expression may be a novel mechanism for the blunted ANP release in heart failure.     

Expression of atrial natriuretic factor (ANF) during Xenopus cardiac development

We have isolated the Xenopus orthologue of the atrial natriuretic factor (ANF) gene. Characterization of embryonic expression indicates that the ANF gene is initially expressed throughout the developing myocardium at the late heart tube stage (about stage 32). This is in contrast to all previously characterized Xenopus cardiac differentiation markers that are first expressed in the cardiogenic plate at approximately stage 27. ANF expression becomes restricted exclusively to the atrium at about stage 47, long after the commencement of beating and the original formation of the atrial and ventricular compartments, but shortly after septation of the single atrium into two distinct atria. Received: 5 May 2000 / Accepted: 3 August 2000     

Structural and functional characterization of recombinant medaka fish alpha-amylase expressed in yeast Pichia pastoris

The medaka fish α-amylase was expressed and purified. The expression systems were constructed using methylotrophic yeast Pichia pastoris, and the recombinant proteins were secreted into the culture medium. Purified recombinant α-amylase exhibited starch hydrolysis activity. The optimal pH, denaturation temperature, and K_M and V_max values were determined; chloride ions were essential for enzyme activity. The purified protein was also crystallized and examined by X-ray crystallography. The structure has the (α/β)₈ barrel fold, as do other known α-amylases, and the overall structure is very similar to the structure of vertebrate (human and pig) α-amylases. A novel expression plasmid was developed. Using this plasmid, high-throughput construction of an expression system by homologous recombination in P. pastoris cells, previously reported for membrane proteins, was successfully applied to the secretory protein.     

Cardiac Atrial Circadian Rhythms in PERIOD2::LUCIFERASE and per1:luc Mice: Amplitude and Phase Responses to Glucocorticoid Signaling and Medium Treatment

Circadian rhythms in cardiac function are apparent in e.g., blood pressure, heart rate, and acute adverse cardiac events. A circadian clock in heart tissue has been identified, but entrainment pathways of this clock are still unclear. We cultured tissues of mice carrying bioluminescence reporters of the core clock genes, period 1 or 2 (per1^luc or PER2^LUC) and compared in vitro responses of atrium to treatment with medium and a synthetic glucocorticoid (dexamethasone [DEX]) to that of the suprachiasmatic nucleus (SCN) and liver. We observed that PER2^LUC, but not per1^luc is rhythmic in atrial tissue, while both per1^luc and PER2^LUC exhibit rhythmicity in other cultured tissues. In contrast to the SCN and liver, both per1^luc and PER2^LUC bioluminescence amplitudes were increased in response to DEX treatment, and the PER2^LUC amplitude response was dependent on the time of treatment. Large phase-shift responses to both medium and DEX treatments were observed in the atrium, and phase responses to medium treatment were not attributed to serum content but the treatment procedure itself. The phase-response curves of atrium to both DEX and medium treatments were found to be different to the liver. Moreover, the time of day of the culturing procedure itself influenced the phase of the circadian clock in each of the cultured tissues, but the magnitude of this response was uniquely large in atrial tissue. The current data describe novel entrainment signals for the atrial circadian clock and specifically highlight entrainment by mechanical treatment, an intriguing observation considering the mechanical nature of cardiac tissue.     

Armadillo-like helical domain containing-4 is dynamically expressed in both the first and second heart fields

Armadillo repeat and Armadillo-like helical domain containing proteins form a large family with diverse and fundamental functions in many eukaryotes. Herein we investigated the spatiotemporal expression pattern of Armadillo-like helical domain containing 4 (or Armh4) as an uncharacterized protein coding mouse gene, within the mouse embryo during the initial stages of heart morphogenesis. We found Armh4 is initially expressed in both first heart field as well as the second heart field progenitors and subsequently within predominantly their cardiomyocyte derivatives. Armh4 expression is initially cardiac-restricted in the developing embryo and is expressed in second heart field subpharyngeal mesoderm prior to cardiomyocyte differentiation, but Armh4 diminishes as the embryonic heart matures into the fetal heart. Armh4 is subsequently expressed in craniofacial structures and neural crest-derived dorsal root and trigeminal ganglia. Whereas lithium chloride-induced stimulation of Wnt/β-catenin signaling elevated Armh4 expression in both second heart field subpharyngeal mesodermal progenitors and outflow tract, right ventricle and atrial cardiomyocytes, neither a systemic loss of Islet-1 nor an absence of cardiac neural crest cells had any effect upon Armh4 expression. These results confirm that Wnt/β-catenin-responsive Armh4 is a useful specific biomarker of the FHF and SHF cardiomyocyte derivatives only.     

Evolutionary Persistence of the Molybdopyranopterin-Containing Sulfite Oxidase Protein Fold

Summary: The importance of molybdoenzymes is exemplified both by the debilitating and fatal human diseases caused by their deficiency and by their persistence throughout evolution. Here, we show that the protein fold of the molybdopyranopterin-containing domain of sulfite oxidase (the SUOX fold) can be found in all three domains of life. Analyses of sequence data and protein structure comparisons (secondary structure matching) show that the SUOX fold is found in enzymes that have quite distinct macromolecular architectures comprising one or more domains and sometimes subsidiary subunits. These are summarized as follows: (i) animal SUOXs that contain an N-terminal cytochrome b₅ domain and an SUOX fold fused to a C-terminal dimerization domain; (ii) plant SUOX that contains an SUOX fold fused to a C-terminal dimerization domain; (iii) the YedY protein from Escherichia coli, which comprises only the SUOX fold; (iv) the sulfite dehydrogenase from Starkeya novella that contains the SUOX fold, a dimerization domain, and an additional c-type cytochrome subunit; and (v) the plant-type nitrate reductases, exemplified by that of Pichia angusta, that contain an N-terminal SUOX fold, a dimerization domain, a cytochrome b₅ domain, and a C-terminal NADH binding flavin adenine dinucleotide-containing domain. We used the primary sequences of the proteins containing an SUOX fold to mine 559 sequences of related proteins. A phylogeny of a nonredundant subset of these sequences was generated, and the resultant clades were categorized by sequence motif analyses in the context of the available protein structures. Based on the motif analyses, cladistics, and domain conservations, we are able to postulate a plausible pathway of SUOX fold enzyme evolution.     

Proteomic analysis of rat atrial secretory granules: a platform for testable hypotheses

The recent development of powerful proteomic tools has enabled investigators to directly examine the population of proteins present in defined biological systems. We report here the first proteomic analysis of atrial secretory granules. Approximately 100 distinct protein components of the atrial secretory granule proteome were detected using subcellular fractionation and one-dimensional SDS-PAGE in conjunction with peptide mass fingerprinting by MALDI-TOF mass spectrometry. Of this number, 61 proteins were clearly identified by high probability data matches and repeated observation. The majority of the proteome was found to be membrane-associated with the most prominent proteins being peptidylglycine alpha-amidating monooxygenase (PAM) and pro-atrial natriuretic peptide (pro-ANP). This proteomic analysis of the rat atrium secretory granule produced an assembly of proteins with a diverse array of reported functions. The identified proteins fall into seven functional categories: (1) granular transport, docking and fusion; (2) signal transduction; (3) calcium-binding/calcium-dependent; (4) cellular architecture/chaperoning; (5) peptide/protein processing; (6) hormone; (7) proton transport. The novel finding of several protein processing enzymes and signal transduction proteins offer new perspectives on how pro-ANP is stored and processed to ANP during release. Accordingly, defining the proteome of the atrial secretory granule provides a framework for the development of new hypotheses that address key mechanisms governing granule function and ANP secretion.     

Localization and characterization of atrial natriuretic factor (ANF)-like peptide in the frog atrium

The distribution of ANF was studied in the heart of the frog (Rana ridibunda) using indirect immunofluorescence. ANF-like immunoreactivity was localized mainly in the right and left atrium, most of cardiocytes being intensively labelled. At the electron microscopic level, all secretory granules present in atrial cardiocytes contained ANF immunoreactive material. Using a specific radioimmunoassay, we found higher concentrations of ANF in the left atrium (208 +/- 25 ng/mg protein) than in the right atrium (120 +/- 16 ng/mg protein) whilst in the rat, the right atrium contains the highest ANF concentration. The concentration of ANF in the ventricle was 10 times lower than in the whole atrium (32 +/- 4 ng/mg protein). Sephadex G-50 gel filtration of atrial extracts showed that ANF-like immunoreactivity eluted in three peaks. Most of the immunoreactivity corresponded to high molecular weight material eluting at the void volume while 20% of the material co-eluted with synthetic (Arg 101-Tyr 126) ANF. These results indicate that frog cardiocytes synthetize a peptide which is immunologically and biochemically related to mammalian ANF.     

Characterization of the third SERK gene in pineapple (Ananas comosus) and analysis of its expression and autophosphorylation activity in vitro

Two somatic embryogenesis receptor-like kinase genes (identified as AcSERK1 and AcSERK2) have previously been characterized from pineapple (Ananas comosus). In this work, we describe the characterization of a third gene (AcSERK3) in this family. AcSERK3 had all the characteristic domains and shared extensive sequence homology with other plant SERKs. AcSERK3 expression was studied by in situ hybridization and quantitative real-time PCR to analyze its function. Intense in situ hybridization signals were observed only in single competent cells and competent cell clusters; no hybridization signal was detected in the subsequent stages of somatic embryogenesis. AcSERK3 was highly expressed in embryogenic callus compared to other organs, e.g., 20–80 fold more than in anther but similar to that of non-embryogenic callus, which was 20–50 fold that of anther. AcSERK3 expression in root was 80 fold higher than in anther and the highest amongst all organs tested. These results indicate that AcSERK3 plays an important role in callus proliferation and root development. His-tagged AcSERK3 protein was successfully expressed and the luminescence of His6-AcSERK3 protein was only ∼5% of that of inactivated AcSERK3 protein and reaction buffer without protein, and 11.3% of that of an extract of host Escherichia coli pET-30a. This finding confirmed that the AcSERK3 fusion protein had autophosphorylation activity.     

Retrieving hidden atrial repolarization waves from standard surface ECGs

Background

This study estimates atrial repolarization activities (Ta waves), which are typically hidden most of the time from body surface electrocardiography when diagnosing cardiovascular diseases. The morphology of Ta waves has been proven to be an important marker for the early sign of inferior injury, such as acute atrial infarction, or arrhythmia, such as atrial fibrillation. However, Ta waves are usually unseen except during conduction system malfunction, such as long QT interval or atrioventricular block. Therefore, justifying heart diseases based on atrial repolarization becomes impossible in sinus rhythm.

Methods

We obtain TMPs in the atrial part of the myocardium which reflects the correct excitation sequence starting from the atrium to the end of the apex.

Results

The resulting TMP shows the hidden atrial part of ECG waves.

Conclusions

This extraction makes many diseases, such as acute atrial infarction or arrhythmia, become easily diagnosed.

     

Differential regulation of the atrial isoforms of the myosin light chains during striated muscle development.

We have isolated a cDNA that encodes the human regulatory myosin light chain isoform predominant in adult atrial muscle. The cDNA contains an open reading frame of 175 amino acids and encodes a hydrophilic protein of a largely helical structure with two potential phosphorylation sites. The protein is different from any other regulatory myosin light chain so far described and is the product of a previously uncharacterized single copy gene. An isoform-specific probe was used to analyze the expression of this isoform in adult muscle and in cardiac and skeletal muscle development in vivo and in vitro. Parallel analysis of the corresponding human alkali myosin light chain (predominant in adult atrium) showed that both isoforms are expressed in early heart development, in both atrium and ventricle. Although the atrial alkali light chain is expressed throughout embryonic striated muscle development, the regulatory myosin light chain was not detected in skeletal myogenesis in vivo or in vitro. Thus the atrial isoforms are not universally or exclusively "paired" and can be independently regulated. We propose that the manner in which these particular isoforms fulfill the functional requirements of the muscle at different developmental times may have direct impact on their regulation.     

Crystal Structure of ATVORF273, a New Fold for a Thermo- and Acido-Stable Protein from the Acidianus Two-Tailed Virus

Acidianus two-tailed virus (ATV) infects crenarchaea of the genus Acidianus living in terrestrial thermal springs at extremely high temperatures and low pH. ATV is a member of the Bicaudaviridae virus family and undergoes extra-cellular development of two tails, a process that is unique in the viral world. To understand this intriguing phenomenon, we have undertaken structural studies of ATV virion proteins and here we present the crystal structure of one of these proteins, ATV. ATV forms tetramers in solution and a molecular envelope is provided for the tetramer, computed from small-angle X-ray scattering (SAXS) data. The crystal structure has properties typical of hyperthermostable proteins, including a relatively high number of salt bridges. However, the protein also exhibits flexible loops and surface pockets. Remarkably, ATV displays a new protein fold, consistent with the absence of homologues of this protein in public sequence databases.     

Pharmacological characterization of the heartbeat in an extant vertebrate ancestor,the Pacific hagfish,Eptatretus stoutii

Pharmacological ion-channel blockers were used to investigate the spontaneous heart rates in Pacific hagfish, Eptatretus stoutii. Zatebradine, a hyperpolarization-activated cyclic nucleotide-gated (HCN) channel blocker, vastly reduced atrial and ventricular contraction rates in a similar concentration-dependent manner, indicating a major role for HCN in setting intrinsic heart rate. When voltage-gated Na⁺ channels were blocked with tetrodotoxin (TTX), atrial contraction rate declined in a dose-dependent manner, but remained faster than ventricular rate even at very high TTX concentrations. This TTX resistance compared with other fish suggests an important role for a TTX-sensitive inactivation-resistant Na⁺ current in atrioventricular conduction and chamber synchrony, and a lesser role in setting intrinsic heart rate. T and L-type calcium channel blockers, nickel and nifedipine respectively, also reduced atrial and ventricular contraction rates, nickel having a larger effect on the atrium. These novel results for hagfish are consistent with intrinsic atrial and ventricular rates being set mostly by HCN, with lesser contributions from other ion channels. We suggest that future electrophysiological studies will reveal that hagfishes, with their ancestral position in the evolution of the vertebrate-type chambered heart, share some but not all features of vertebrate intrinsic heart rate control.     

Potassium channel blocking 1,2-bis(aryl)ethane-1,2-diamines active as antiarrhythmic agents

Atrial fibrillation (AF) is a major cause of stroke, heart failure, sudden death and cardiovascular morbidity. The Kv1.5 potassium channel conducts the IKur current and has been demonstrated to be predominantly expressed in atrial versus ventricular tissue. Blockade of Kv1.5 has been proven to be an effective approach to restoring and maintaining sinus rhythm in preclinical models of AF. In the clinical setting, however, the therapeutic value of this approach remains an open question. Herein, we present synthesis and optimization of a novel series of 1,2-bis(aryl)ethane-1,2-diamines with selectivity for Kv1.5 over other potassium ion channels. The effective refractory period in the right atrium (RAERP) in a rabbit PD model was investigated for a selection of potent and selective compounds with balanced DMPK properties. The most advanced compound (10) showed nanomolar potency in blocking Kv1.5 in human atrial myocytes and based on the PD data, the estimated dose to man is 700?mg/day. As previously reported, 10 efficiently converted AF to sinus rhythm in a dog disease model.