Beating Rat Heart Cells Research Articles

Stimulation and Homologous Desensitization of Calcitonin Gene-Related Peptide Receptors in Cultured Beating Rat Heart Cells*

Addition of calcitonin gene-related peptide (CGRP), 1 X 10(-7) M, to cultured neonatal rat heart cells resulted in rapid increases in beating rate and cellular concentrations of cAMP. Calcitonin (1 X 10(-7) M), in contrast, had no significant effect on heart cell beating rate or cAMP content. CGRP-stimulated increases in heart cell cAMP content were rapid, transient, dose dependent, and potentiated by isobutyl-methylxanthine (1 X 10(-4) M). Half-maximal increases in heart cell cAMP content occurred at 1 X 10(-8) M CGRP. Heart cell adenylate cyclase responses to CGRP were desensitized in a rapid (i.e. within 5 min) and dose-dependent manner by prior exposure to CGRP. Complete and half-maximal desensitization of heart cells to CGRP occurred at 1 X 10(-8) and 3 X 10(-10) M CGRP, respectively. Desensitization of heart cells to CGRP did not modify the cAMP response of heart cells to beta-adrenergic agonist stimulation, and beta-adrenergic agonist desensitization of heart cells did not modify responses to CGRP. Heart cell cAMP responses to CGRP were additive to those of the beta-adrenergic agonist isoproterenol and occurred in the presence of beta-adrenergic blockade. These observations demonstrate that CGRP exerts specific and potent agonist actions in cardiac myocytes and that regulation of myocardial responses to CGRP may occur by mechanisms involving increases in cAMP and receptor desensitization.

Verapamil reduces glucose and fatty acid metabolism of beating and nonbeating rat heart cells.

The effects of various dosages of verapamil on glucose or fatty acid utilization by beating or nonbeating rat heart myocytes in tissue culture were determined. Myocytes were incubated with verapamil and either D-6-14C glucose or 1-14C palmitic acid as substrate. After incubation the subsequently generated 14CO2 was captured with hyamine hydroxide and the equivalent oxygen values were calculated. Low doses of verapamil (50 ng/ml) treatment produced a 52% reduction in myocyte glucose utilization and a 16% reduction in fatty acid utilization that appeared to be independent of its effect on myocyte contractile rate since these effects were evident in both beating and nonbeating myocytes. In addition, verapamil treatment caused differences in the myocyte handling of substrate. Verapamil (50 ng/ml) lowered cellular accumulation of glucose by 21% compared to controls. Contrary to glucose, myocyte concentrations of palmitic acid were significantly increased by 117% relative to controls in verapamil treated cultures. These results suggest that verapamil may have a direct effect on basal heart cell metabolism in a way that is unrelated to myocyte contractile activity. In addition, verapamil may interfere with glucose membrane transport.

Coxsackie B-2 virus infection in rat beating heart cell culture

Beating rat heart cultures were prepared in vitro and infected with Coxsackie B-2 virus. The cells were evaluated in the post-infected period for changes in cardiac enzymes, alterations in beating frequency and cytotoxicity as measured by chromium 51 ( 51Cr) release. The cardiac enzymes, lactate dehydrogenase (LDH) and aspartate aminotransferase (AST) were measured in infected and uninfected controls over a period of 120 h. Enzyme levels in the infected cells remained essentially the same for the first 42 h as compared to the controls. At this time, the LDH levels increased rapidly reaching 116 ± 24.8 U/l while the controls remained at 46.9 ± 9.7 U/l. Aspartate aminotransferase levels increased at a slower rate and obtained a level of 104 ± 20.2 U/l compared to 66.6 ± 13.2 U/l in the control. Visual evidence of cellular damage as measured by decreased beating frequencies and the appearance of cytopathic effect was first noted at 42 h post-infection. Complete loss of cardiac beats and maximal viral cytopathic effect occurred at 96 h post-infection. Cardiac cellular damage as measured by cytotoxicity assay was found to parallel those changes seen in cardiac enzymes. No significant changes in cytotoxicity were observed for the first 24 h; however, at 48 h increased release of 51Cr was noted and visual evidence of viral replication also was present. The cardiac enzyme changes noted in beating rat heart cells appear to be similar to those changes reported in patients with viral-induced myocardial disease. This would suggest that the use of beating rat heart cultures can be a useful model in the study of viral-induced cardiac diseases, and may give insight into the use of anti-viral medications for the management of these disorders.

The effect of jaundiced sera and bile salts on cultured beating rat heart cells.

Jaundiced serum from common bile duct ligated rats, added to cultured heart cells, decreased the beating rate, caused an early cessation of beating and production of higher levels of lactate in the media. Deoxycholate and cholate are the main bile acids in jaundiced serum; deoxycholate caused similar effects, which suggests that it is the toxic substance responsible for heart function alterations seen in patients with severe jaundice.

A high-performance liquid chromatographic method to measure 32P incorporation into phosphorylated metabolites in cultured cells

Orthophosphate is rapidly transported into cultured cells and subsequently incorporated into numerous compounds. A high-performance liquid chromatographic method that enables the measurement of 32 P i incorporation into acid-soluble metabolites in cultured cells treated with exogenous 32 P i is described. Baseline resolution and quantitative recovery of 12 ribonucleotides are accomplished in less than 75 min. In cultured, beating rat heart cells, the concentration and extent of labeling by 32 P i of most phosphorylated metabolites were unchanged in cells treated with the anesthetic halothane (2-bromo-2-chloro-1,1,1-trifluoroethane). The method is generally applicable to the investigation of phosphate transport and incorporation by numerous cell types under various experimental conditions.

Das Vorkommen kreuzreaktiver Antigene in Herzmuskelgewebe und Streptococcus pyogenes IV. Zytotoxischer Effekt von Lymphozyten auf Primärkulturen von Herzmuskelzellen nach In-vivo-Senibilisierung mit Streptokokken der Gruppe A

The cytotoxic effect of rat lymphocytes in vivo sensitized to group A streptococci, rat heart tissue or streptococcal M proteins was investigated in primary cultures of beating rat heart cells. Cytotoxicity was found in 75 to 82% of heart cell cultures in contact with lymphocytes sensitized to streptococcal whole cells or lysates and in 100% of those exposed to lymphocytes sensitized to rat heart tissue. Lymphocytes sensitized to M protein exert a cytopathic effect only in 8 and 12% cultures, respectively, and to a much more limited extent. Controls were affected in 6%. The cytotoxicity test seems to be useful to examine the presence of cross-reactive antigens within streptococcal vaccines.

Electron microscopical studies of the effect of antiheart antibodies and complement on beating heart cells in culture

Ultrastructural changes in cultured beating rat heart cells, treated with specific antibodies and complement, have been described. The addition of complement after the specific antibody, resulted in an immediate arrest of the beating, while in the control group the cells continued to beat until the end of the experiment, i.e. for 60 min. The degree of cellular damage did correlate with the time of exposure to the antibody and complement, while the percentage of damaged cells did increase. This could be related to the difference in the number of antibody molecules attaching themselves to antigenic sites on the cell membrane. It is assumed that the damage to the beating heart cells was due to defective control of the cell membrane osmolarity due to interference in Na-K ATPase activity.

Influence of variations in pericellular oxygen tension on individual cell growth, muscle-characteristic proteins, and lactate dehydrogenase isoenzyme heart cells.

Primary cell cultures from neonatal rat ventricles were continuously exposed for 7 days in a modified roller apparatus to defined pericellular oxygen tension varying from 0.6 to 600 mm Hg. 5-Fluorodeoxyuridine was added to the medium to prevent over-growth of muscle cells by nonmuscle cells. A pericellular pO2 of 600 mm Hg was lethal. The range of about 15 to 150 mm Hg was favorable, as indicated by increases in total and muscle-characteristic proteins. Between the 2nd and 8th day of cultivation at a pO2 of 38 mm Hg, myosin content per cell increased 3.2-fold and creatine kinase activity 2.5-fold. At 0.6 mm Hg, myosin content increased only 1.3-fold and there was no increase in creatine kinase activity. The rate of myosin synthesis was diminished at this low pO2. ATP level and beating rate at 0.6 mm Hg did not differ from values at 38 mm Hg. The isoenzyme pattern of lactate dehydrogenase remained unchanged during cultivation at 38 mm Hg, whereas at 0.6 mm Hg it shifted towards an M-type pattern. These experiments suggest that neonatal rat heart cells maintained in vitro can adapt themselves to low oxygen tensions.

Toxic effects of DMSO on cultured beating heart cells at temperatures above zero

Toxic influences of DMSO were studied on spontaneously beating rat heart cells in tissue culture. Injurious effects of DMSO on the contractility and on the individual cell survival were investigated at three different temperatures, +4 ° C, +24 ° C, and + 37 ° C. As indicated by the 50% levels of irreversible damage it may be concluded that osmotic stress during the addition and the removal of DMSO should be avoided carefully at +4 ° C. With slow introduction and slow removal of DMSO optimal survival may be obtained up to 4.7 m DMSO at +4 ° C and at +24 ° C. Although at +37 ° C osmotic stress becomes less damaging, optimal survival can only be obtained at lower concentrations (up to 3.5 m depending on the incubation program). At +37 ° C the more specific toxic properties of DMSO are the limiting factor. At +24 ° C slow removal of DMSO instead of sudden removal at the end of an incubation period improves the survival considerably. At +24 ° C a good compromise between damage by osmotic shock and by other more specific toxic properties can be obtained.

The influence of hypertonic solutions on cultured beating rat heart cells

Hypertonic stress caused by sucrose and sorbitol followed by reincubation in isotonic medium causes irreversible damage to synchronously beating heart cells. These results can be explained by a “Minimum Cell Volume” theory as proposed by Meryman. During exposure to hypertonic NaCl solutions damage to the contractility depends on the incubation time. Damage to the individual cell survival seems to be less dependent on the incubation period.

The Cardiac Auto-Immune System

Using rabbit anti-rabbit heart auto-antibodies, cultures of dissociated beating rat heart cells were examined for the presence of heart auto-antigens by immunofluorescence. Several patterns of staining of myocardial cells were observed. One type of auto-antibody revealed crisp myofibrillar cross-striations, consisting of wide bright segments often split by a thin dark band. Auto-antibody producing this pattern was potent for auto-antigen B. Rabbit anti-rabbit heart auto-antibodies rich in the auto-antigen D systems produced a granular staining in the myocardial cells, while an auto-antibody reactive with all five of the cardiac auto-antigens showed both granular and myofibrillar cross-striations. All the heart auto-antibody preparations reacted with cultured neonatal rat skeletal muscle cells to show diffuse cytoplasmic fluorescence and fine myofibrils. In all cases, rabbit anti-rabbit heart auto-antibodies failed to stain significantly the non-contractile cells present in the tissue cultures (‘endotheliod’, fibroblast, etc.). Sarcolemmal staining was not a prominent feature of these reactions. When the rabbit anti-rabbit heart auto-antibodies were exposed to living beating rat heart cells, no staining was observed, indicating the absence of these antigens on the cell surface.

The effect of adenosine on cardiac cells in culture

Chronotropic effect of 5 × 10 −6 m to 5 × 10 −5 m-adenosine on beating rat heart cells in culture was demonstrated. Concomitant with the increased rate of rhythmic contractions was a 50% increase in the intracellular ATP concentration, a net synthesis of c. 15 nmol ATP/mg protein. This net change in concentration was a result of a new high steady state of ATP synthesis and not of a transphosphorylation from phosphorylcreatine, as the latter remained at control levels. The rhythmic contractions and the increased ATP synthesis in the presence of adenosine were inhibited by oligomycin + 2-deoxyglucose and 1-fluoro-2,4-dinitrobenzene The excitability of the cardiac cell membrane correlates with a continuous synthesis of ATP.

α-Aminoisobutyric acid uptake by cultured beating heart cells

1. 1. The uptake of the amino acid analog, α-aminoisobutyric acid (AIB) by cultured beating rat heart cells has been investigated, taking advantage of a culture technique enabling to obtain 80–90 % myoblasts. 2. 2. AIB was found to be transported and accumulated within the myoblasts by a process requiring energy, K + and Na +, as evidenced by the marked drop in AIB uptake observed in myoblasts incubated with oligomycin, or in the absence of Na + or K + in the medium. 3. 3. The observed decrease in net AIB uptake brought about by the lack of either Na + or K + in the medium appears, however, to be related more to drastic changes in ionic gradients than to a modulation of the Na +-K + pump activity, as the myoblasts were very insensitive to ouabain. 4. 4. Glycolysis is likely to be of considerable importance for optimal AIB accumulation by the myoblasts. Thus, iodoacetate markedly inhibited this process; glucose completely overcame the inhibitory effect of oligomycin on net AIB uptake, an action of glucose that could be prevented by further addition of 2-deoxyglucose. 5. 5. When added alone to the incubation medium, insulin failed to alter AIB uptake. However, under conditions in which the energy supply was limited ( e.g. cells treated with oligomycin) and AIB accumulation consequently decreased, the hormone brought the uptake of the amino acid back towards the normal level provided glucose (not pyruvate) was present in the medium. This suggests that insulin acts on the amino acid concentrative process mainly via its well-substantiated stimulatory effect on the transmembrane transport of glucose.

The effect of insulin on the glycolysis and glycogen content of beating rat heart cells in the primary culture.

An pulsierenden Rattenherzzellen in der Primarkultur wurde die Insulinwirkung auf die Glykolyse und den Glykogengehalt untersucht.

Positive chronotropic response of cultured isolated rat heart cells to N6,2′- O-dibutyryl-3′,5′-adenosine monophosphate

In cultures of spontaneously and rhythmically beating rat heart cells which respond to adrenaline with an increase in the rate of beat, N 6-2′- O-dibutyryl-3′,5′-cyclic AMP mimics this effect. The positive chronotropic action of this compound is concentration-dependent, the lowest effective concentration being approximately 10 −6 m, and it is potentiated by caffeine. Cyclic AMP itself, butyrate, 5′-AMP, ADP and ATP, even in relatively high concentrations, were without effect on the rate of beat of the cultured heart cells. An activating effect of positive chronotropic concentrations of adrenaline and dibutyrul cyclic AMP on the phosphorylase system of the cultured beating heart cells could not be demonstrated, since glycogen phosphorylase in these cells was present entirely as 5′-AMP-independent enzyme. The observations on the stimulating influence of dibutyryl cyclic AMP upon the automacity of the cultured heart cells suggest, in conjunction with other evidence reported in the literature, that cyclic AMP is an intermediate in the pathway of the positive chronotropic action of adrenaline on cardiac pacemaker cells.

In vitro production of chromosomal lesions with an argon laser microbeam.

THE development of the laser microbeam has made available an additional tool for selective probing of cellular organelles. Individual mitochondria have been studied successfully by Bessis and his colleagues, using a ruby laser microbeam1. The large mitochondria of beating rat heart cells have been extensively investigated using an argon laser microbeam arrangement (unpublished results of D. E. R. and N. Gamaleja). Another organelle which it would be important to manipulate is the nucleus with its nucleolar and chromosomal components. It seemed reasonable to assume that, given a compatible in vitro culture system and laser microbeam apparatus, lesions could be placed on predetermined sites of selected chromosomes.

Studies in vitro on single beating rat heart cells : IX. The restoration of beating by serum lipids and fatty acids

1. 1. Heart cells incubated in lipid-deficient media lose their ability to beat. 2. 2. The presence of lipid-free serum proteins partially maintains growth but not beating. 3. 3. The presence of serum lipids maintains beating but not growth. 4. 4. The addition of lipid-free serum and serum lipids may reconstitute whole serum in its ability to maintain growth and beating. 5. 5. Lipid-free albumin maintains cells without growth or beating. 6. 6. The addition of serum lipids or fatty acids to cells, maintained either in lipid-free serum proteins or lipid-free albumin, and which have lost their beating, restores the ability to beat.

The effect of lipids on enzyme levels in beating rat heart cells

Non-dividing beating rat heart cells, incubated in a lipid free medium, showed a decrease in malic dehydrogenase and Ca-ATPase and an increase in glucose 6-phosphate dehydrogenase. These changes were associated with a cessation of beating. Serum lipids, added to the medium, prevented the enzyme changes and maintained the beating. Serum lipids also restored the beating and reversed the enzyme changes which were caused by the lipid deficiency.

In Vitro studies on single beating rat heart cells : VI. Electron micorscopic studies of single cells

The ultrastructure of single beating heart cells, isolated from newborn rats by trypsinization and grown in semisynthetic media using a modified feeder layer technique, was investigated. In many of the cells myofibrils with A, I, and Z bands were seen. In all beating cells more or less well developed myofilaments were seen. Varying developmental stages occurred in cultures of the same age. Well developed mitochondria and Golgi bodies were also present. Ribosomes were found linearly arranged parallel to the myofilaments organized in what appeared to be a helix. Polysomes, scattered filaments, and Z material seem to arise in the periphery of the cell. The relation of these polysomes to the synthesis of myofilaments and the development of myofibrils is discussed.

In vitro studies on single beating rat heart cells : II. Intercellular communication

Summary 1. Rat heart cells separated by trypsin and grown in vitro continue to beat. 2. Cells beating at an independent rate become synchronous upon physical contact, indicating that the communication of rate of beating is through protoplasmic contact. 3. The rate of beating for the synchronous net of muscle cells is determined by the fastest beating cell. 4. Despite the superimposed rate, each cell retains potentially its own inherent rate of beating.