Adult Atria Research Articles

Aging alters the force-frequency relationship and toxicity of oxidative stress in rabbit heart

Adult (6 months) and senescent (> 5 years) rabbit atria were studied under conditions known to increase cytoplasmic calcium (increased frequency of contraction and oxidative stress). At a contraction frequency of 1/sec, cardiac relaxation (90% relaxation time) was similar in senescent and adult atria but at a frequency of 2 or 3/sec, relaxation was significantly slower in senescent preparations (P < 0.05). Additional experiments indicated that H 2O 2 (500 μM), a powerful oxidant, increased resting force and decreased developed force (DF) much more rapidly in senescent than adult atria; the maximum decrease in DF, however, was less in senescent preparations (adult = 81 ± 6% and senescent = 42 ± 27% of pre-H 2O 2 values; P < 0.05). Age-related differences in effects of H 2O 2 did not result simply from a decreased ability of senescent hearts to detoxify an oxidative stress by the glutathione pathway. Both basal glutathione (GSH) concentrations and the H 2O 2-mediated decreases in GSH were similar in adult and senescent ventricular preparations, as were activities of glutathione peroxidase and glutathione reductase. These observations suggest that interventions known to increase cytoplasmic calcium can amplify age-related impairments of cardiac relaxation through mechanisms that may be independent of the glutathione pathway.

Expression of G proteins in rat cardiac myocytes: effect of KCl depolarization

As a first step in understanding the functioning of hormonal signaling pathways in the heart, the G protein composition of neonatal and adult rat hearts was determined by immunoblotting analysis. Neonatal rat cardiac myocytes and nonmuscle heart cells contained Gs alpha 52, Gi alpha 1,3, and Gi alpha 2 with little or no Gs alpha 45 or Go alpha 39. Interestingly, Go alpha 39 accumulated fourfold in cardiac myocytes between days 1 and 6 of culture. Atrial membranes from adult rat hearts had approximately equal amounts of the 45- and 52-kDa forms of Gs alpha, whereas adult ventricles had predominantly Gs alpha 45. In addition, adult atria contained relatively more Gi alpha 1,3, Gi alpha 2, and Go alpha 39 than adult ventricle. Moreover, the increase in Go alpha 39 observed in cardiac myocytes cultured for 6 days could be prevented by culturing cells in medium containing 50 mM KCl. The observed differences in G protein expression between cell types and between contracting and KCl-depolarized cardiac myocytes may provide a system to investigate the function of various G proteins and to study regulation of their expression.

Perinatal expression of the atrial natriuretic factor gene in rat cardiac tissue.

The expression of the atrial natriuretic factor (ANF) gene was examined in the perinatal rat ventricle. The late fetal ventricle (day -3 relative to parturition) demonstrated a modest level of ANF gene expression. This expression increased at the time of birth and peaked on day +1 after birth. Atrial ANF immunoreactivity (irANF) increased gradually during the postpartum period (day +1 to 2-3 wk of age), while ventricular irANF as well as ventricular ANF mRNA levels fell over the same interval. This decrease in ANF gene expression resulted from a decrease in activity of the same major transcription start site employed in the adult atria and ventricle. Plasma irANF levels were very high in the neonatal period and decreased over the ensuing 2-3 wk, a profile similar to that seen for ventricular ANF gene expression. In situ hybridization histochemistry revealed that, while adult rat ventricular ANF mRNA was predominantly confined to the subendocardial layer, neonatal ventricles expressed the ANF gene throughout approximately 50% of the inner wall thickness. These findings suggest that the ANF gene is responding to regulatory stimuli that appear at or near the time of parturition and dissipate in the early neonatal period.

Differential tissue expression and developmental regulation of guanine nucleotide binding regulatory proteins and their messenger RNAs in rat heart.

The expression and developmental regulation of the alpha and beta subunits of the guanine nucleotide binding regulatory proteins, Gi and Go, were examined in rat atria and ventricles. Protein levels were determined by quantitative immunoblot analysis using affinity purified monospecific antibodies. Northern blot and dot blot analyses were used to characterize and quantitate relative amounts of mRNA encoding these G protein subunits. The concentrations of Go alpha, Gi alpha, and beta subunit protein were found to be greater in adult atrial than in adult ventricular membranes (5.2-, 1.5-, and 2.8-fold, respectively). A corresponding 3.4-fold difference in Go alpha mRNA level was also observed, as well as a 1.3-fold difference in Gi alpha-3 mRNA level. No difference was seen between the amount of beta, Gi alpha-1, Gi alpha-2 mRNA in adult atria and adult ventricles. Comparison of neonatal and adult tissues revealed a developmental decrease in ventricular Gi alpha protein and Gi alpha-2 mRNA levels (70 and 47%, respectively). Developmental decreases were also observed in the amount of mRNA encoding beta and Go alpha in ventricles (47 and 61%, respectively), and beta and Gi alpha-2 in atria (40 and 36%, respectively), while a developmental increase in atrial Gi alpha-3 mRNA levels was observed (57%). These results demonstrate differences in the expression of G protein subunits in rat atria and ventricles, as well as regulation of the levels of these subunits during cardiac development.

Alkali myosin light chains in man are encoded by a multigene family that includes the adult skeletal muscle, the embryonic or atrial, and nonsarcomeric isoforms

A set of cDNA clones coding for alkali myosin light chains (AMLC) was isolated from fetal human skeletal muscle. Nucleotide sequence analysis and RNA expression patterns of individual clones revealed related sequences corresponding to (i) fast fiber type MLC ! and MLC 3 ; (ii) the embryonic MLC that is also expressed in fetal ventricle and adult atrium (MLC emb); and (iii) a nonsarcomeric MLC isoform that is found in all nonmuscle cell types and smooth muscle. The AMLC gene family in man comprises unique copies for MLC 1, MLC 3 and MLC emb, and multiple copies for the nonsarcomeric MLC genes. The gene coding for MLC 1 and MLC 3 is located on human chromosome 2.

Developmental patterns of expression and coexpression of myosin heavy chains in atria and ventricles of the avian heart

Monoclonal antibodies (mAbs), electrophoresis, immunoblotting, and immunohistochemistry were used to determine the molecular properties of cardiac myosin heavy chain (MHC) isoforms and the regions of the developing chicken heart in which they were expressed. Adult atria expressed three electrophoretically distinct MHCs that reacted specifically with mAbs F18, F59, or S58. During embryonic Days 2–4, when the atrial and ventricular chambers are forming, MHCs that reacted with mAbs F18, F59, or S58 were expressed in both the atria and ventricles. The atria continued to express MHCs that reacted with mAbs F18, F59, or S58 at all stages of development and in the adult. In the ventricles, expression of the MHCs reacting with these mAbs was found to be developmentally regulated. By embryonic Day 16, MHC(s) reacting with mAb F18 had disappeared from the developing ventricles, whereas MHCs reacting with S58 and F59 continued to be expressed throughout the ventricles. As development continued, MHC(s) reacting with S58 in the ventricle became restricted to expression in only the ventricular conducting system. MHC(s) reacting with F59 were expressed in both the ventricular myocytes and the ventricular conducting system throughout development and in the adult. Thus, in contrast to the embryonic chicken heart where at least three MHC isoforms were expressed in both the atria and ventricles, we found in the adult chicken heart that— at a minimum—three MHC isoforms were expressed in the atria, two MHC isoforms were expressed in the ventricular conducting system, and one MHC isoform in the ventricular myocardium. MHC isoform expression in the developing avian heart appears to be more complex than previously recognized.

Tension reactivation and potentiation in guinea pig atrium: the effects of isoprenaline, calcium and rate changes.

The process of tension repriming and the phenomenon of tension potentiation after premature stimulation (post-extrasystolic potentiation, PESP) were studied in the adult guinea pig atrium. The following results were obtained. (i) Reducing extracellular calcium, [Ca]o, to 50% of normal did not significantly change the rate of tension repriming. However, in the presence of 5 microM isoprenaline (which greatly speeded up repriming) the same reduction in [Ca]o slowed down the repriming process. (ii) Increases in the rate of stimulation enhanced the rate of tension repriming in a control medium, but this rate-dependence was absent in the presence of isoprenaline. (iii) Isoprenaline (20 microM) abolished PESP. A reduction in [Ca]o or the addition of verapamil (still with isoprenaline) partly restored tension potentiation. In neonatal guinea pig atria, a large PESP was evident, which was only slightly reduced by isoprenaline. These results are interpreted as reflecting changes induced by isoprenaline in the degree of filling of sarcoplasmic reticulum (SR) stores with calcium, and in the rate of calcium recycling between uptake and release sites within the SR network. The large PESP found in the neonate, and its relative insensitivity to isoprenaline was interpreted as reflecting a scarcity of SR. This implies that tension potentiation may also reflect changes in sarcolemmal calcium currents.

Effects of Postnatal Maturation on Postrest Potentiation in Isolated Rabbit Atria

Ultrastructural changes in cardiac sarcoplasmic reticulum (SR) have been reported during postnatal development of the mammalian heart, but the functional significance of these observations has not been well characterized. Calcium release from SR in intact myocardial preparations was determined by the contractile characteristics of postrest contractions. Isometric tension and the maximum rate of tension development of the first contraction following intervals of electromechanical quiescence (rest) were related to steady-state tension and maximum rate of tension development during contraction at constant frequency (1.0 Hz) in isolated left atrial strips from newborn (1-7 days), immature (14-21 days), and adult (more than 6 months) rabbits. The first postrest contraction was increased as a function of the rest interval rate of tension development, defined as postrest potentiation, in all three age groups and reached a maximum value at rest intervals of more than 20 s. Tension developed by the first contraction following a 60-s rest interval was potentiated less in newborn and immature atria than in adult atria at an extracellular calcium concentration ([Ca]e) of 2.5 mM, an age-related difference most marked in the immature. Ryanodine (5.0 X 10(-9) M), a putative blocker of calcium release from cardiac SR, abolished postrest potentiation providing evidence that calcium release from SR is the predominant determinant of the postrest contraction. Postrest tension in atria from the immature rabbit heart was significantly increased both in absolute terms and relative to steady-state tension following stabilization under conditions which increase intracellular [Ca] [( Ca]i), i.e. increasing [Ca]e, increasing tonicity, or decreasing extracellular sodium concentration ([Na]e).(ABSTRACT TRUNCATED AT 250 WORDS)

Atrial natriuretic polypeptide in the fetal rat: ontogeny and characterization.

The presence of immunoreactive atrial natriuretic polypeptide (ANP) has been demonstrated in fetal atria early in gestation but further definition of fetal ANP has not been reported. To characterize the principal molecular forms of fetal ANP and to compare fetal ANP to that of the adult of the same species, we extracted the atria of pregnant adult and 20-day fetal rats, the hearts of 14-day fetuses, and intact 12-day fetuses in 1 M acetic acid. Tissue collected from littermates was pooled. We measured ANP by radioimmunoassay before and after gel filtration on Sephadex G-75 in each group. ANP concentrations (means +/- 1 SD) in ng/mg protein and ng/animal were 1296 +/- 505 and 7707 +/- 1877 in adult atria (n = 17), 174 +/- 44 and 62 +/- 13 in 20-day fetal atria (n = 7), and 33 +/- 5.3 and 3.7 +/- 0.9 in 14-day fetal hearts (n = 6), respectively. Acid extracts from intact 12-day fetuses did not dilute in parallel to the standard curve; therefore, concentrations of ANP for the 12-day fetuses are not reported. ANP concentration rose from the 20-day fetus to the adult (p less than 0.0001). The major species of ANP eluting from the Sephadex column had an apparent molecular weight of 16 K in all groups. We conclude: 1) ANP is present in the fetus shortly after the completion of organogenesis; 2) 16 K ANP is the principal intracardiac species in the fetus and the adult; and 3) the existence of ANP soon after cardiac development suggests a possible role for ANP in fetal blood pressure and sodium and water homeostasis.

Atriopeptin distribution in the developing rat heart.

The embryonic distribution of atriopeptin (atrial natriuretic factor) in the Sprague-Dawley rat heart was mapped by immunoperoxidase staining of embryonic and neonatal hearts using rabbit antiserum to atriopeptigen purified from adult rat atrium. During the period of cardiac septation (days 14 and 16), immune serum reacted strongly with myocardial cytoplasmic granules in two sites: the inner cell layer along the cephalic curvature of the atria and the trabeculae of the incompletely divided ventricles. The youngest hearts studied (gestational day 11) displayed only nonspecific diffuse peroxidase reactivity within blood cells, indistinguishable from control sections incubated with normal rabbit serum. One week following birth, intense anti-atriopeptin reactivity was widely distributed through both atria. In addition, immunoreactive cytoplasmic granules were found at several sites in the ventricular myocardium. Along the fiber tracts of the concentric layers of the ventricular walls and interventricular septum, scattered granular foci were seen between nuclei of contiguous elongated myocytes. Positive staining was also seen within the papillary muscles and trabeculae carnae, regions shown by Alcian blue/periodic acid-Schiff base staining of sister sections to be relatively rich in glycogen. These patterns of antibody reactivity suggest the coupling of early atriopeptin secretory activity with developing cardiac function.

The conducting tissue in the adult chicken atria. A histological and immunohistochemical analysis.

A three-dimensional reconstruction from serial sections of adult chicken heart was made to verify whether Purkinje cells, that can be recognized by a number of well-known histological criteria, form specialized tracts in the adult chicken atria. This reconstruction revealed a loosely arranged network of Purkinje cells connecting the two atria. This network has not been described before. No tracts could be detected between the sinoatrial and the atrioventricular nodes. These atrial Purkinje cells express the atrial and ventricular myosin isoform, as determined by the use of monoclonal antibodies that were prepared against atrial and ventricular myosin isoform, respectively. Some atrial myocytes that are topographically closely related to the Purkinje cells and that cannot be distinguished from the surrounding myocytes with conventional histological criteria, express, apart from the atrial myosin isoform, also the ventricular myosin isoform. The similar expression pattern of these two cell types and their close topographical relationship suggest the presence of a more elaborate system specialized in conduction than the well-known conductive system found with conventional histological techniques.

Isolation of cardiac myosin light‐chain isotypes by chromatofocusing

Cardiac myosin light chain isotypes have been resolved using chromatofocusing, a new preparative column chromatographic technique. The method relies on production of narrow-range, shallow and stable pH gradients using ion-exchange resins and buffers with even buffering capacity over the required pH range. Light chains were resolved in order of decreasing isoelectric point in the pH range 5.2-4.5. Gradients of delta pH = 0.004-0.006/ml elution volume were achieved which were capable of resolving light chains with isoelectric point differences of only 0.03. Analytical isoelectric focusing of light chains in polyacrylamide gels could be used to predict the results of preparative chromatofocusing for method development. Chromatofocusing was capable of resolving human and bovine cardiac light chain 1 and 2 subunits, atrial (ALC) and ventricular (VLC) light chain isotypes and homologous VLC-2 and VLC-2* light chains. The technique was used to purify and resolve the human foetal ventricular light chain 1 (FLC-1) from adult ventricular light chain 1 (VLC-1) present in foetal ventricles and the atrial light chain 1 (ALC-1) in adult atria. Comparative peptide mapping studies and amino acid analyses were carried out on FLC-1 and ALC-1. No differences were detected between FLC-1 and ALC-1 using three different proteases and amino acid compositions were similar with the exception of glycine content. The studies indicate that FLC-1 and ALC-1 are homologous, and possibly identical, light chains. Comparison of human FLC-1/ALC-1 with VLC-1 suggested marked structural and chemical differences in these light chain isotypes, in particular in the contents of methionine, proline, lysine and alanine residues. Differences in the contents of these residues were also apparent in the corresponding bovine atrial and ventricular light chains [Wikman-Coffelt, J. & Srivastava, S. (1979) FEBS Lett. 106, 207-212]. The latter three residues are known to be rich in the N-termini of cardiac and skeletal light chain 1 isotypes, an area that has been implicated in actin binding, suggesting that atrial and ventricular light chains may differ functionally in this region.

Characterization of a myosin heavy chain in the conductive system of the adult and developing chicken heart.

A monoclonal antibody (anterior latissimus dorsi 58 [ALD58]; antimyosin heavy chain, MHC) directed against myosin from slow tonic muscle was found to react specifically with the striated muscle cells of the conductive system in the adult chicken heart. This monoclonal antibody was used to study the expression of myosin in the conductive system of the adult and developing heart. Using immunofluorescence microscopy with ALD58, muscle cells of the conductive system were demonstrated in both the atria and ventricles of the adult heart as previously shown by Sartore et al. (Sartore, S., S. Pierobon-Bormioli, and S. Schiafinno, 1978, Nature (Lond.), 274: 82-83). Radioactive myosin from adult atria and ventricles was precipitated with ALD58 and subjected to limited proteolysis and subsequent peptide mapping. Peptide maps of ALD58 reactive myosin from atria and ventricles were very similar, if not identical, but differed from peptide maps of ordinary atrial and ventricular myosin. The same antibody was used to study cardiac myogenesis in the chick embryo. When ALD58 was reacted with myosin isolated from atria and ventricles at selected stages of development in radioimmunoassays, reactivity was not observed until the last week of embryonic life (greater than 15 d of egg incubation). Thereafter concomitant and progressively increased reactivity was observed in atrial and ventricular preparations. Also, no ALD58 positive cells were observed in immunofluorescence studies of embryonic hearts until 17 d of egg incubation. Primary cell cultures of embryonic hearts also proved to be negative for this antibody. This study demonstrates that an epitope recognized by ALD58 associated with an antimyosin heavy chain of striated muscle cells of the adult heart conductive system is absent or present in only small amounts in the early embryonic heart.

Immunohistochemical and immunocytochemical localization of atrial natriuretic polypeptide in human adult and fetal hearts.

The localization of atrial natriuretic polypeptide (ANP) in human adult and fetal hearts was investigated by an immunoperoxidase procedure using anti-α-human ANP antiserum. Light microscopically, the reaction product for ANP was observed in atrial muscle cells and was well confined within both the atrium and atrial septum with the exception of the specialized conducting system of adult heart. No prominent difference in intensity of immunostaining was observed in any part of the atrium. Intracytoplasmic immunoreaction product for ANP was finely granular and generally concentrated at paranuclear sarcoplasm. Electron immunogold stain for ANP of the human adult atrium was also carried out. Ultrastructurally, gold particles were demonstrated in atrial specific granules. Immunoreactive-ANP was also demonstrated in all atria of fetuses at 7, 11, 12, 13, 16 and 17 weeks of gestation. The distribution of the reaction products was similar to those of the adult heart, and there was a tendency of increased staining intensity for ANP with gestational age.

Atrial specific granules of the human auricle in embryogenesis, tissue culture, and hypertrophy.

The ultrastructure of atrial specific granules of the human auricle has been studied in myoblasts of 8-12 week embryos, in myoblasts cultured for 15 days, and in cells of the hypertrophied juvenile and adult atrium. The granules of young myoblasts and those of cultured cells are fairly evenly distributed throughout the sarcoplasm. Myocytes of the hypertrophied juvenile auricle show structural characteristics indicative of granule formation. Structural evidence of exocytosis has not been observed in either of the tissues studied. In the cultured and the hypertrophied tissue the granules are structurally well preserved even in areas with extensive intracellular lysis.