Adult Rat Myocardium Research Articles

Distribution of myosin isozymes within single cardiac cells. An immunohistochemical study.

Isozymes of myosin have been localized with respect to individual cardiac myocytes in hearts from 3-week-old, adult controls, and adult hypophysectomized rats, and in cultured cardiac cells. For this purpose, affinity-purified antibodies reacting specifically with the heavy chains of each of the two major myosin isozymes of adult rat heart, V1 and V3, were used. The distribution of the two isomyosins was determined by double immuno-labeling of the same cell, V1 myosins being revealed by rhodamine and V3 myosins by fluorescein. A procedure is described which allows optimum immunological visualization of the myosin filaments of rod-shaped isolated myocytes. It was found that the response of the cardiac cells to the two antimyosins varied depending on the state of the animal. In 3-week-old rats, all cells were stained with the anti-V1, and almost none with the anti-V3 myosin. In the hypophysectomized animals, on the contrary, all cells were stained with the anti-V3 and none with the anti-V1. A mixed pattern of reactivity was observed in adult controls since 50% of the cells reacted with the anti-V1, 10% with the anti-V3, and 40% with both antibodies. In the latter case, the distributions of V1 and V3 reactivities were homogeneous throughout the cell, and absolutely superimposable. The same double reactivity and homogeneous repartition were observed in cultured cells. These findings indicate that myocytes from adult rat myocardium are heterogeneous in terms of their isomyosins content and show for the first time that two isomyosins can coexist and be equally distributed in one cardiac cell. These observations are relevant to the regulation of individual heart cell contractility.

Ontogeny of α 1-adrenergic receptors in the rat myocardium: Effects of hypothyroidism

The effects of propylthiouracil (PTU) treatment on α 1- and β-adrenergic receptors in neonatal and adult rat myocardium were examined with the α 1-selective antagonist [ 3H]prazosin and β-antagonist (−)-[ 3H]dihydroalprenolol ((−)-[ 3H]DHA). Pre and postnatal PTU treatment (congenital hypothyroid state) decreased ventricular size and protein content at 15 and at 28 days (P < 0.01). Similarly PTU treatment after maturation (acquired hypothyroid state) is associated with decreased ventricular size and protein content (P < 0.01). PTU treatment did not alter ventricular DNA content at any age examined as compared to euthyroid controls. Specific [ 3H]prazosin binding to ventricular membrane increased with advancing postnatal age in both congenital hypothyroid pups and controls, reaching peak receptor density at 15 days of postnatal age (106 ± 8 vs. 151 ± 7 fmol·mg −1 protein). The density at 15 and 28 days was however significantly lower in hypothyroid pups (P < 0.01, P < 0.02). The effect of congenital hypothyroidism on (−)-[ 3H]DHA binding was more marked which significantly decreased from the first postnatal day. Acquired hypothyroidism is also associated with a decreased density of α 1- and β-adrenergic receptors in rat myocardium. We conclude that α 1- and β-adrenergic receptors are modulated by thyroid hormone in development well as mature stages of the rat.

Electrophoretic analysis of multiple forms of rat cardiac myosin: Effects of hypophysectomy and thyroxine replacement

Electrophoretic analysis in pyrophosphate gels of intact myosin of adult rat myocardium revealed the presence of five distinct components, two in atrial myosin (A 1, A 2) and three in ventricular myosin (V 1, V 2, V 3). Analysis of Ca 2+-activated myosin ATPase activity in the gels revealed that A 1, A 2 and V 1 had about the same specific activity; V 3 had the lowest activity, while that of V 2 was intermediate. At 3 weeks of age, ventricular myosin was exclusively V 1, there being a slow age-dependent shift in myosin distribution toward the adult pattern. Hypophysectomy of juvenile rats caused a shift towards V 3, which by 45 days after operation accounted for 90% of total myosin. This shift was prevented by daily administration of 5 μg of thyroxine. When chronically hypophysectomized rats were similarly treated, there was a rapid shift of myosin components towards V 1. These changes in distribution of myosin components were associated with appropriate changes in Ca 2+-activated ATPase activity of electrophoretically purified ventricular myosin as measured in the gel. Sodium dodecyl sulphate gel analysis of purified myosin showed little or no contaminants. For both V 1 and V 3 rich myosins, the ratio of the two ventricular light chains (V-LC 1, V-LC 2) and the ratio of light chains to heavy chains are consistent with the model that each intact molecule contains two each of V-LC 1 and V-LC 2. Sodium dodecyl sulphate electrophoresis of purified atrial myosin revealed two types of light chains: A-LC 1 (mol. wt 27 000, not resolved from V-LC 1) and A-LC 2 (mol. wt 22 000), the latter being distinct from V-LC 2.

Studies of sarcoplasmic reticulum function and contraction duration in young adult and aged rat myocardium

Kinetic measurements of Ca +2 uptake in microsomal fractions from rat myocardium demonstrated significantly lower rates of oxalate-facilitated accumulation in preparations from aged (24 to 25 month) hearts as compared to those from young adult (6 to 8 month) hearts. The per cent decline in transport activity in microsomes from aged hearts varied with Ca 2+ concentration decreasing from 57% at 0.33 μ m Ca 2+ to 24% at 1.21 μ m Ca 2+. Double-reciprocal plots of the dependence of the velocity of accumulation on Ca 2+ concentration showed upward curvature in both age groups indicating the presence of multiple Ca 2+ binding sites. Mechanical studies using muscles isolated from the same hearts used to prepare sarcoplasmic reticulum demonstrated prolonged contraction duration in aged myocardium in agreement with previous findings. The lower in vitro rates of Ca 2+ accumulation in aged microsomes suggest a possible biochemical mechanism to account for the observed increase in the time-course of cardiac relaxation.

Surface characteristics of cells isolated from adult rat myocardium

The general surface morphology of single cells isolated enzymatically from adult rat myocardium has been examined using scanning electron microscopy. Many of the dispersed cells are approximately cylindrical in shape and show transverse ridges and longitudinal grooves on their surfaces. T-tubules appear in regular arrays, repeat at intervals consistent with sarcomere lengths for contracted cells and envelop myofibrils in a cuff-like manner. T-tubule openings are both ovoid and circular in shape. Intercalated disk regions are convoluted in appearance and exhibit morphology consistent with the general findings observed in whole tissue. These data confirm the usefulness of isolated myocytes as a model for morphological studies on the complete sarcolemmal surface of individual muscle cells derived from myocardial tissue.

Separation of muscle and non-muscle cells from adult rat myocardium: An application to the study of RNA polymerase

Non-muscle cells, closely adherent to myocytes, can be demonstrated by nuclear staining techniques in isolated myocardial cells. The method for isolating myocardial muscle and non-muscle cells that we have described significantly reduces cross-contamination. This procedure makes it possible, for the first time, to measure RNA synthesis, as nuclear RNA polymerase activity, in separated muscle and non-muscle cells. Biochemical reactions can now be studied simultaneously in two entirely different cell types from the same organ under similar conditions.

QUANTITATIVE ANALYSIS OF ULTRASTRUCTURAL CHANGES IN DEVELOPING RAT CARDIAC MUSCLE DURING NORMAL GROWTH AND DURING ACUTE VOLUME LOAD

1. In comparison with adult rats, the myocardial cells of young post-natal rats contain more ribosomes, glycogen particles and roughsurfaced endoplasmic reticulums, but fewer mitochondria and myofibrils. The mean volume percentages of mitochondria both atria and both ventricles, those of myofibrils are 44 to 48%, and those of glycogen particles are 14 to 18%. 2. In comparison with young post-natal rats, the myocardial cells of 7-day-old rats contain better-developed mitochondria and myofibrils, but fewer ribosomes, glycogen particles and rough-surfaced endoplasmic reticulums. 3. The volume percentages of mitochondria, myofibrils or glycogen particles in 3-month-old rats are almost the same as those of adult rats. Namely, the mean total areas occupied in the sarcoplasm by mitochondria in the four chambers are 32 to 36%, those by myofibrils are 53 to 58%, and those by glycogen particles are 4 to 8%. 4. Thus, for the three main intracellular components; mitochondria myofibrils, and glycogen particles; there are no significant differnces among the mean contents in the four chambers at any age. 5. In spite of abrupt circulatory changes at birth, both damages of myofibrils and intracellular organs and intracellular edema are seldom seen in the myocardium of young post-natal rats. Such shows that the young post-natal rat cardiac muscle has sufficient tolerance to these natural loads. 6. Mitosis was found in the cardiac muscle cells of an 8-day-old rat and cell proliferation after birth was confirmed. 7. Throughout the developmental periods, the minimum diameters of the nuclei in both atria and both ventricles did not greatly alter to become significantly different. 8. The cell diameter of the left atrium of 0-day-old rats was significantly smaller than that of the right atrium, right ventricle or left ventricle. The cell diameters of the right atrium and right ventricle were not significantly altered until the 7th day after birth, but that of the left atrium increased markedly for the same period, and then, became equal to that of the right atrium or right ventricle. Further, the cell diameter of the left ventricle increased markedly. After that period, the cell diameters of both atria and both ventricles increased prominently with increasing age. The increase was more prominent in the ventricular than in the atrial myocardiums. 9. Mitochondrial destruction and intracellular edema were found in the myocardium of young post-natal rats after volume load by blood equalling the whole blood volume. In the myocardium of adult rats during the same volume load, however, the above damages were only slight, and numerous enlarged mitochondria with increased number of cristae occupied a larger intracellular space. These results show that the tolerance of young post-natal cardiac muscle is poorer than that of adult cardiac muscle.

Isoprenaline stimulation of cyclic AMP production by isolated cells from adult rat myocardium

Summary When 1.25 × 10−6 mol/l isoprenaline is added to suspensions of adult rat myocardial cells in calcium-free bicarbonate buffer. there is a 1.6-fold increase above control of cyclic AMP levels 30 seconds after drug addition. The phosphodiesterase inhibitor methylisobutylxanthine (MIX) at 0.5 mmol/l increases the control level of cAMP and also the level of stimulation to 3-fold. When MIX is present, Ca++ (1 mmol/l) has no effect on either control or stimulated cAMP, whereas omission of Mg++ results in a lowering of control cAMP, with the levels after stimulation unchanged. Propranolol at 8.45 × 10−6 mol/I aholishes stimulation by isoprenaline.

A rapid technique for the isolation and purification of adult cardiac muscle cells having respiratory control and a tolerance to calcium

A rapid technique is described for the isolation of muscle cells from adult rat myocardium using in vitro perfusion with calcium-free bicarbonate buffer containing crude collagenase. Under optimum conditions, 5 × 10 6 cells per g tissue are obtained and the suspension may be purified to contain 70% intact cells. The complete procedure is rapid and isolated myocytes beat, exclude vital stains, have conventional sub-cellular morphology, show tight respiratory coupling and also have a tolerance to external calcium not found in cells isolated by other perfusion techniques. This represents a significant advance in the development of an isolated cell model for the study of myocardial mechanisms.

Histochemical and morphological observations on rat myocardium after exercise.

Effects of seven levels of chronic physical activity on the metabolic and morphologic characteristics of left ventricular myocardium of adult male albino rats were investigated.