Activation of contractility and occurrence of aftercontractions in Ca2+ and Sr2+-solutions during the postnatal development of the rat heart

Activation of isometric contractile force and induction of aftercontractions by different extracellular Ca- and Sr-concentrations during the early postnatal development of the rat heart were studied. In the neonatal (1-15 days) rat heart activation of contractility by [Ca]0 and [Sr]0 were similar when a dose-response curve for Ca was determined before Sr, but if the experiment was performed in the reverse order of the dose-response curve for Sr was shifted to the left. In either case the maximal developed tension was about the same. In the adult (3-5 months) rat heart [Ca]0 higher than 4 mM was inhibitory, whereas contractile force increased up to 16 mM [Sr]0 without any signs of force depression. The dose-response curve for Sr was shifted to the right compared to that of Ca and the maximal developed tension was clearly higher in Sr-solution than in Ca-solution. Aftercontractions appeared for the first time on the 13th and 24th postnatal days for 16 mM [Ca]0 and [Sr]0, respectively. In the adult rat ventricle lower Ca concentrations were needed to induce aftercontractions compared to Sr. Based upon these observations it is suggested that the appearance of aftercontractions during the third postnatal week of rat heart development is due to the maturation of intracellular Ca stores which become available for contractile activation by the development of the T-system. The absence of a negative inotropic effect in elevated Sr concentrations may be due to the slowing down of the transport processes of the sarcoplasmic reticulum (SR) by Sr and to the greater ability of the SR to store Sr over Ca.     

Mechanical responses of developing Fisher rat heart. Effects of steroid hormone

Age-dependent changes in the mechanical responses of developing Fisher rat heart during the first three postnatal weeks were studied in relation to the hypothesis that the abnormality observed in the mechanical responses of the rat heart might be calcium related. Therefore the effect of frequency of stimulation as well as the response to calcium, epinephrine and ouabain on hearts of untreated and cortisol-treated rats was compared. The positive force-frequency response observed in fetal rat heart reverted to a highly negative response by the 12th to 14th postnatal day. The biphasic mechanical responses directly paralleled reported changes in circulating glucocorticoid levels in developing rat. The force-frequency response was maximally negative when the circulating levels of glucocorticoids were lowest. The reversion of the negative force-frequency responses coincided with a gradual increase reported in the circulating levels of glucocorticoids. The negative force-frequency response was absent in the cortisol-treated developing rat heart and a definite positive pattern was observed as the rats developed. A high sensitivity to free calcium concentration, seen in control fetal and and newborn hearts, diminished after the second postnatal week. By the third postnatal week, the sensitivity to high extracellular calcium concentrations was significantly reduced. The sensitivity to calcium persisted in the cortisol-treated hearts during the 3 postnatal weeks. Cortisol-treated hearts were more responsive to epinephrine than controls. The abbreviation of time to peak tension, a hallmark of the catecholamine effect, was observed at a younger age in the cortisol-treated hearts. Cortisol-treated hearts were more responsive to the inotropic effects of ouabain than controls. The possible involvement of glucocorticoids in the control of calcium handling elements of the myocardium is discussed.     

Postnatal development of beta-adrenergic response in ventricular muscle of the rat heart

In adult mammalian, heart responses to beta- and alpha-adrenergic stimulation are different: the beta-type effect exhibits a larger increase of relaxation than of contraction, while the alpha-stimulation has no selective influence on relaxation. The present results show that the effect of isoprenaline (ISO) on the neonatal rat heart during the 1st postnatal week is not a typical beta-effect in that the relaxant influence of beta-stimulation is lacking. During the 2nd and 3rd postnatal weeks the typical beta-response, with improved relaxation, gradually appears. The absence of the typical beta-effect is not caused by the lack of beta-receptors or cAMP-dependent phosphorylation reactions because in other respects, the positive inotropic effect of ISO is well developed at the moment of birth. In addition to these qualitative changes, also prominent quantitative changes occurred in the ISO response. The dose-response curves were shifted to the right with advancing age, suggesting reduced beta-agonist potency of the maturing tissue. The developed tension (Tmax) abruptly increased between the 12th and 17th postnatal days and then steeply declined during the next 2 weeks. Changes in Tmax correlated fairly well with the general ability of the tissue to generate extra force, as expressed by rest-dependent potentiation of twitch. However, during the 2nd postnatal week cardiac tissue seemed to be subsensitive to ISO, since all contractile parameters except T'max were depressed. The results suggest that the postnatal changes in beta-response are primarily determined by alternations in the electromechanical coupling process of the developing tissue, and less by the proper adrenergic mechanisms.     

Activation of contractility and sarcolemmal Ca2+-ATPase by Ca2+ during postnatal development of the rat heart

The effects of 0.25-16.0 mM Ca2+ on the contractile force of isolated ventricular strips and sarcolemmal Ca2+-ATPase activity during postnatal development of the rat heart were studied. The half maximal concentrations for contractile activation of ventricular strips were 0.76 and 5.59 mM Ca2+ for adult and 3-day-old rats, respectively. The sensitivity towards Ca2+ began to change from newborn type to that of adult rat 2 weeks after birth and was almost completed after 4 weeks. No significant differences were found in half maximal activation of Ca2+-ATPase by Ca2+ between different age groups. Activation of contractility and Ca2+-ATPase by Ca2+ were linearly related in 30-day-old and adult rats but not in 3- and 10-day-old rats. The observed sensitivity change towards extracellular Ca2+ for contractile activation is suggested to be due to the development of transverse tubular system and sarcoplasmic reticulum during the first 4 weeks of postnatal development.     

Electrical properties of developing rat heart. Effects of dexamethasone

Action potentials recorded from perinatal rat ventricles exhibited a plateau (phase 2), followed by a rapid repolarization characteristics of all mammalian ventricular cells. Within the second postnatal week, a number of distinct changes occurred in the contour of action potentials. An early slow depolarization, at the foot of the action potential, preceded the beginning of phase zero. The early slow depolarization was observed until day 12 and disappeared by day 13. A second slow depolarization occurred during the terminal phase of the rapid upstroke of the action potential, persisted through day 13 and disappeared by day 14. On day 12, what had been a homogeneous contour of action potentials seen during the first week converted into a heterogeneous contour. Occasionally, action potentials similar to those recorded from Purkinje fibres in adult heart were recorded from hearts as young as 12 days. By day 14, signs of a spike (the hallmark of action potentials from adult heart) were apparent in some fibres. Treatment of newborn rats with dexamethasone on the second day after birth prevented the disappearance of the second slow depolarization. In adult and aged rat hearts, dexamethasone treatment induced a slow depolarization and a plateau in the region of overshoot. In view of the time-dependent change of the second slow depolarization it is suggested that this phase of the action potential is influenced by the levels of circulating glucocorticoid in developing heart and by changes in calcium sensitivity observed in this species. Heterogeneity of action potentials observed on day 12 postnatal may precede structural differentiation of myofilaments.     

Cellular energy metabolism during fetal development. IV. Fatty acid activation, acyl transfer and fatty acid oxidation during development of the chick and rat

We have investigated developmental profiles of ATP-dependent palmityl-CoA synthetase, acetyl-CoA synthetase, palmitylcarnitine transferase, and fatty acid oxidation in heart and liver of developing chicks and rats. Palmityl-CoA synthetase activity of rat liver and heart homogenates increased 6- to 10-fold during the first postnatal week. Chick embryo heart activity peaked between 13 and 16 days of development. The activity of embryonic chick livers was bimodal with highest activity seen at 7 and 16 days of development. Posthatching values were approximately 50–75% of the peak embryonic levels. Acetyl-CoA synthetase activity of rat liver and heart homogenates was low but also showed developmental increases following birth. Acetyl-CoA synthetase activity of chick embryonic hearts was greatest at 16 days of development. Palmitylcarnitine transferase activity of rat liver and heart homogenates showed a striking increase during the first week of life. Chick heart activity was similar to that observed for palmityl-CoA synthetase with a peak between 13 and 16 days of embryonic development. Coincident with the postnatal rise in fatty acid activation and palmitylcarnitine transferase activity in developing rats, the oxidation of palmityl-CoA plus carnitine and of palmitylcarnitine increased from barely measurable levels at birth to adult levels by 30 days of age. The increases that we observe probably relate to changes in the specific activity of the enzymes as well as to an increase in the absolute number of mitochondria during development.     

Effects of caffeine on the mechanical properties of developing rat heart ventricles

The effects of caffeine on isometric contractions of right-ventricular strips during the postnatal development of the rat heart were studied. Caffeine (2-10 mM) had a positive inotropic effect on ventricular strips of 3-15-days-old rats but a negative inotropic effect on the muscles of rats older than 22 days. Rest-twitch potentiation was most prominent in the muscles of 3-15-days-old rats but weakened clearly after that age. The potentiation was eliminated by 5 and 10 mM caffeine. An abrupt increase in frequency from 0.2 to 2 Hz caused a positive tension-staircase in 3-15-days-old rats but a negative staircase in older rats, the latter effect being eliminated by caffeine. It is suggested that the observed changes during the third postnatal week are due to a shift from extracellular to intracellular Ca2+ as the main source of this cation.     

Vasoactive intestinal polypeptide in rat heart atria: the effect of hyperthyroidism

The effects of transient and sustained hyperthyroidism on vasoactive intestinal polypeptide-like immunoreactivity (VIP-LI) levels were studied in the heart atria of developing and adult rats. Newborn rats were divided into 5 groups. Neo-T animals were treated with thyroxine (T4) during postnatal days 1-8 and sacrificed at the age of 60 days. Neo-S rats were treated with T4 during postnatal days 1-60 and sacrificed one day later. Adult-1 and Adult-2 animals received T4 during days 52-60 and were sacrificed 5-6 days and 1 day later, respectively. Control animals were injected with saline. VIP-LI concentrations were determined in extracts from the left and right atria separately. In Neo-S and Adult-2 rats, spontaneous heart rate, the weight of both atria and total T4 serum levels were significantly enhanced, while their body weight was decreased. The ratio atria weight to body weight was significantly increased in all groups except for Adult-1 animals. Hyperthyroidism led to a significant decrease in VIP-LI levels in both atria of Neo-S and Neo-T rats. Hyperthyroidism induced in adult rats also decreased VIP-LI levels in both atria. However, this change was only transient. In conclusion, our data have provided new evidence that hyperthyroidism induced during the early neonatal period interferes with the development of VIP-ergic innervation in rat atria. The period of the first few postnatal days seems to be essential for this effect, since VIP-LI concentrations in 60-day-old animals did not significantly differ between Neo-S and Neo-T atria.     

The use of fluorescent antimyosin and DNA labeling for the estimation of the myoblast and myocyte population of primary rat heart cell cultures

Cell division in heart muscle cells progressively ceases during the development of the rat heart, leading to an adult stage with muscle cells incapable of cell division. We have quantitatively determined the number of dividing and nondividing heart muscle cells in cultures derived from different stages of the developing rat heart with the use of ³HTdR continuous labeling and fluorescent antimyosin staining. The cultures were derived from 14 and 17 day postcoital (dPC) rat embryos and from 1 and 4 day postnatal (dPN) rats. The percent nondividing cells increased with development and the age of the postnatal rat. The percent nondividing cells in 14 dPC equalled 21%, 17 dPC equalled 25%, 1 dPN equalled 44%, and 4 dPN equalled 60%. This method for the quantitative determination of dividing and nondividing cells in the developing rat heart provides a model that is useful for the study of the mechanism of the loss of cell division capacity.     

Developmental expression patterns of connexin26 and -30 in the rat cochlea

Connexin proteins form transmembranous gap junction channels that connect adjacent cells. Connexin26 and connexin30 have been previously shown to be strongly expressed in the inner ear of adult rats and to be mainly colocalized. Because intercellular connections by gap junction proteins are crucial for maturation of different tissues, we investigated the developmental expression of connexin26 and connexin30 in pre- and postnatal rats using immunocytochemistry. In the rat otocyst, staining for connexin26 as well as for connexin30 appeared at the 17th day of gestation. However, at this stage, expression of connexin30 was low and restricted to the neurosensory epithelium. Beginning from the 3rd postnatal day connexin26 and -30 were expressed with highest immunoreaction in the spiral limbus, the neurosensory epithelium, and between the stria vascularis and the spiral ligament. Beginning from postnatal day 12 the staining pattern resembled that of adult animals, with additional strong staining between all fibrocytes of the spiral ligament. Double labeling experiments demonstrated strongest colocalization of both connexins between the stria vascularis and the spiral ligament. These results demonstrate that development of the cochlear gap junction system precedes the functional maturation of the rat inner ear, which takes place between the 2nd and 3rd postnatal week. In the cochlea of a 22-week-old human embryo, connexin26 and connexin30 could be detected in the lateral wall, suggesting that both connexins also play a crucial role in function of the human inner ear.     

Thyroid hormone differentially regulates cellular development in neonatal rat heart and kidney.

The role of thyroid hormone in the control of cardiac and renal cell development was examined in neonatal rats made hyperthyroid by administration of triiodothyronine (T3, 0.1 mg/kg s.c. on postnatal days 1-5) or hypothyroid by administration of propylthiouracil (PTU, 20 mg/kg s.c. given to dams on gestational day 17 through postnatal day 5 and to pups on postnatal days 1-5). Indices of total cell number (total DNA per tissue), cell packing density (DNA per g tissue), and relative cell size (protein/DNA ratio) were evaluated from birth through young adulthood. PTU administration led to primary shortfalls in cell number that were of similar magnitude in both tissues, but persisted somewhat longer in the kidney than in the heart. Deficits in cell packing density and cell size in the hypothyroid animals were secondary to the effect on cell number, displaying smaller magnitudes of effect and a lag in appearance and disappearance of the deficits compared to that for total DNA; indeed, the phase in which tissues were restoring their cell numbers was accompanied by increased cell packing density, reflecting a more rapid restitution of cell numbers than tissue weight or cell size. In contrast to the relatively similar effects of PTU on developing cardiac and renal cells, the effects of T3 were selective for the heart. Although T3 caused general growth impairment, it evoked marked cardiac overgrowth that was accompanied by a striking increase in cell number and a small increase in cell size. The cardiac hyperplasia is unique to the developing animal, as post-replicative heart cells in adult animals show only hypertrophy in response to thyroid hormone.(ABSTRACT TRUNCATED AT 250 WORDS)     

Developmental expression patterns of connexin26 and ‐30 in the rat cochlea

Connexin proteins form transmembranous gap junction channels that connect adjacent cells. Connexin26 and connexin30 have been previously shown to be strongly expressed in the inner ear of adult rats and to be mainly colocalized. Because intercellular connections by gap junction proteins are crucial for maturation of different tissues, we investigated the developmental expression of connexin26 and connexin30 in pre‐ and postnatal rats using immunocytochemistry. In the rat otocyst, staining for connexin26 as well as for connexin30 appeared at the 17th day of gestation. However, at this stage, expression of connexin30 was low and restricted to the neurosensory epithelium. Beginning from the 3rd postnatal day connexin26 and ‐30 were expressed with highest immunoreaction in the spiral limbus, the neurosensory epithelium, and between the stria vascularis and the spiral ligament. Beginning from postnatal day 12 the staining pattern resembled that of adult animals, with additional strong staining between all fibrocytes of the spiral ligament. Double labeling experiments demonstrated strongest colocalization of both connexins between the stria vascularis and the spiral ligament. These results demonstrate that development of the cochlear gap junction system precedes the functional maturation of the rat inner ear, which takes place between the 2nd and 3rd postnatal week. In the cochlea of a 22‐week‐old human embryo, connexin26 and connexin30 could be detected in the lateral wall, suggesting that both connexins also play a crucial role in function of the human inner ear. Dev. Genet. 25:306–311, 1999. © 1999 Wiley‐Liss, Inc.     

Developmental changes in sugar responses of the chorda tympani nerve in preweanling rats

To clarify developmental changes in the gustatory system of the rat, integrated taste responses from the chorda tympani (CT) nerve were recorded and analyzed at different postnatal ages. The response magnitude was calculated relative to the response to the standard, 0.1 M NH4Cl. Even at 1 week of age, the CT responded well to all tested 0.1 M chloride salts (NH4Cl, NaCl, LiCl, KCl, RbCl and CsCl). The responses to 0.1 M NaCl and LiCl increased with increasing age of the rat while response magnitudes to KCl, RbCl and CsCl did not change up to 8 weeks. At 1 week, the integrated response pattern was quite similar to that in adult rats for NaCl, HCl and quinine hydrochloride (QHCl). The concentration-response functions for NaCl, HCl, QHCl and sucrose at 2 weeks were essentially the same as those at 8 weeks. These results suggest that taste buds in the 2-week-old rat are functionally mature for the detection of the four basic taste stimuli. The relative magnitude of the responses to the various sugars was smaller at 1 week compared to the adult rat and reached a maximum at weeks 3-4, then decreased gradually with age. Among the six sugars, sucrose was the most effective followed by lactose. From weeks 1-4, the magnitude of the integrated taste response to fructose was smaller than that to lactose except at 3 weeks of age. Maltose, galactose and glucose were less potent stimuli than the other sugars tested. The response magnitude to lactose at 4 weeks had decreased compared to that for the other sugars. Taste responses to the sugars in preweanling and adult rats were not cross-adapted by the individual sugars. These results suggest that after 1 week of age during postnatal development in the rat, taste information from the CT rapidly increases in its importance for feeding behavior.     

Changes in force and calcium sensitivity in the developing avian heart.

The aim of this study was to characterize the development of the contractile properties of intact and chemically skinned muscle from chicken heart and to compare these characteristics with those of developing mammalian heart reported by others. Small trabeculae were dissected from left ventricles of Arbor Acre chickens between embryonic day 7 and young adulthood (7 weeks post-hatching). At all ages, increasing extracellular calcium (0.45-3.6 mM) progressively increased twitch force of electrically stimulated trabeculae. Twitch force at 1.8 mM extracellular calcium, normalized to cross-sectional area, increased to a maximum at 1 day post-hatching, remained constant through 3 weeks post-hatching, but then decreased at 7 weeks post-hatching. The maximal calcium-activated force of trabeculae chemically skinned with Triton X-100 detergent increased to a maximum 2 days before the time of hatching and was not significantly changed up to 7 weeks post-hatching. Over the ages studied, average twitch force in 1.8 mM calcium was between 26 and 66% of maximal calcium-activated force after skinning, suggesting that the contractile apparatus is not fully activated during the twitch in normal Ringer. In skinned trabeculae, the calcium sensitivity of the contractile apparatus was higher in the embryo than in the young adult. These age-dependent changes in calcium sensitivity are correlated with isoform switching in troponin T. A decrease in pH from 7.0 to 6.5 decreased the calcium sensitivity of the contractile apparatus to a greater degree in skinned trabeculae from young adult hearts than in those from embryonic hearts. This change in susceptibility to acidosis is temporally associated with isoform switching in troponin I.(ABSTRACT TRUNCATED AT 250 WORDS)     

Action of thyroxine on the cytochrome content in the brain and liver mitochondria during the postnatal development of rats

The content of cytochromes c + c1, b and a in brain and liver mitochondria in 7-day-old rats reaches the level seen in adult animals. Administration of L-T4 in a dose of 0.7 micrograms/g rat bw for 4 days before sacrifice results in activation of cytochrome synthesis in both test organs within the first week of the suckling rats' life. On the 20th day of the postnatal period the effect of T4 is seen only in the liver while the brain tissue turns out indifferent to the thyroid hormone. Thus, T4 activates cytochrome biosynthesis in brain mitochondria during the first week of the rats' life, that leads to the acceleration of the functional activity and higher differentiation of the developing brain mitochondria.     

Effect of lipid diet on mitochondrial palmitoyl-l-carnitine oxidation in kidney at postnatal development.

Oxygen consumption (VO2) and beta-hydroxyacyl-CoA dehydrogenase (beta OAC) activity were measured in isolated mitochondria of developing rat kidney from late fetal to adult age. In the presence of palmitoyl-L-carnitine, VO2 consumption was higher in suckling than in adult rats while beta OAC activity rose during the postnatal period and declined after weaning. During postnatal development, the high level of mitochondrial fatty acid oxidation was linked to the high level of fatty acid supply and any change in lipid diet altered mitochondrial fatty acid oxidation. By contrast at adult age, a high fat diet did not change either mitochondrial fatty acid oxidation or beta OAC activity measured in two nephron structures (PCT and mTAL). Dietary lipids seem to play an important role in the evolution of mitochondrial fatty acid oxidation in developing rat kidney.     

Parallel Postnatal Development of Choline Acetyltransferase Activity and Muscarinic Acetylcholine Receptors in the Rat Olfactory Bulb

The development of cholinergic synapses in the rat olfactory bulb was investigated by measuring changes in the activity of choline acetyltransferase (ChAT; EC 2.3.1.6.), a presynaptic cholinergic marker, and in the concentration of muscarinic receptors, components of cholinoceptive membranes. Three biochemical properties of the muscarinic system also were examined for possible differentiation: ligand binding, molecular weight, and isoelectric point. Receptors from embryonic (day 18), neonatal (postnatal day 3), and adult rat olfactory bulbs exhibited identical complex binding (nH = 0.45) of the agonist carbachol. For each age, the relative proportions of high-affinity (Ki approximately equal to 1.0 microM) and low-affinity (Ki approximately equal to 100 microM) binding states were 60% and 40%, respectively. The antagonist pirenzepine also bound to high-affinity (Ki approximately equal to 0.15 microM, RH approximately equal to 70%) and low-affinity (Ki approximately equal to 2.0 microM, RL approximately equal to 30%) sites in neonatal and adult rats. Sodium dodecyl sulfate/urea-polyacrylamide gel electrophoresis of [3H]propylbenzilylcholine mustard-labeled receptors from neonatal and adult rats showed a single electrophoretic form with an apparent molecular weight of 65,000. In contrast, analytical isoelectric focusing indicated high pI (4.50) and low pI (4.00) receptor forms were present. Neonatal rats contained approximately equal proportions of the two receptor forms, whereas adult rats contained mainly the low pI form, indicating that molecular alteration of the receptor population had occurred during development. Comparison of postnatal changes in acetylcholine receptors and ChAT activity showed a striking correlation between the development of cholinergic terminals and muscarinic receptors. Throughout the first postnatal week, ChAT activity remained at 5% of adult levels; activity began to rise on postnatal day 6 and gradually reached adult levels (56 +/- 4 mumol of [3H]acetylcholine/h/g) during the fourth week. Similarly, muscarinic receptor concentration was low (30-50 fmol/mg) throughout the first week, began to rise at postnatal day 7; and reached 90% of adult levels (317 +/- 17 fmol/mg) by the fourth week. In contrast, there was little increase in the concentration of nicotinic acetylcholine receptors (30 fmol/mg) during this period. The parallel postnatal development of ChAT activity and muscarinic receptors suggests the existence of factors that couple the differentiation of presynaptic cholinergic terminals and postsynaptic cholinoceptive elements.     

Respiratory Cardiac Arrhythmia in Postnatal Ontogenesis of Rats

Development of the respiratory cardiac arrhythmia and the role of parasympathetic nervous system in its origin have been studied in rats aged from 4–6 days to 6 months of life. In rat pups of the first week of life, small fluctuations of cardiac rhythm were observed with the frequency close to fluctuations of respiratory rhythm. However, at this age they had neither regular character nor clear connection with phases of the respiratory cycle. On the 2–3rd week the amplitude of fluctuations rose and their association with respiration was established; however, unlike the respiratory arrhythmia observed in other animals and human, in rat pups there was deceleration but not acceleration of heart beating. By to the 6-week age the respiratory arrhythmia reached the maximal values, then its amplitude began to decrease. Bilateral transection of the vagus nerves in rat pups did not cause reduction of the respiratory arrhythmia. Thus, in rats the central influences on the heart can be transduced by bypassing the system of vagus nerves.