Intrinsic factors influencing the maintenance of contractile embryonic heart cells in vitro. 3. The effect of inoculum level

Seven-day embryonic heart cells are tested for their ability to condition their own medium by comparing cell responses at various inoculum levels. The data show that contractile activity, spreading on glass, and survival increase as inoculum level rises. The data also show that the cell death rate is inversely proportional to the rate at which cell spreading and contractile activity increase.     

Synchrony of cell spreading and contraction force as phagocytes engulf large pathogens

A simple micromechanical method has been used to directly measure the force of contraction in single mammalian phagocytes (blood granulocytes) during engulfment of large yeast pathogens. Both the time course of cell spreading over the yeast particle and increase in cell body contractile force were quantitated at three temperatures in the range of 23-35 degrees C. The surprising feature of the phagocyte response was that engulfment and cell body contraction occurred in a serial sequence: i.e., the phagocyte spread rapidly over the particle at a steady rate with no detectable cell body contraction; when spreading stopped, contraction force in the cell body then rose steadily to a plateau level that remained stationary until the next sequence of spreading and contraction. Both spreading and contraction exhibited abrupt start/stop kinetics. Also impressive, the cell contraction force stimulated by phagocytosis was quite large (approximately 10(-8) N)-two orders of magnitude larger than the force necessary to deform passive phagocytes to the same extent. If distributed uniformly over the cell cross section, the contraction force is equivalent to an average contractile stress of approximately 10(3) N/m2 (0.01 Atm). These physical measurements in situ set critical requirements for the mechanism of force generation in granulocytes, imply that a major increase in network cross-linking accompanies build- up in contractile force and that subsequent network dissolution is necessary for locomotion.     

Attachment, spreading and growth of VERO cells on microcarriers for the optimization of large scale cultures

The VERO cell attachment, spreading and growth were measured as a function of the substrate and temperature used for cell cultivation, the presence of fetal calf serum (FCS) in the medium and the initial cell inoculum used for cultivation on MCs. The data show that the cell attachment kinetics were comparable at RT or 37v°C, a higher rate of cell attachment occurred to MCs and the presence of FCS inhibited the cell attachment to glass or plastic but not to MCs. The cell spreading, in general higher at 37v°C, was dependent on the presence of FCS, comparable on glass or plastic substrate and lower on MCs. The spread of VERO cells over MCs was fully dependent on the presence of FCS and decreases progressively with a delayed addition of FCS into the medium. The cell detachment by trypsin was slower from MCs and the cells recovered showed lower viability and reattachment. Better results of detachment, viability and reattachment were obtained by treatment with the trypsin at pH of 8 instead of 7. The lower was the number of cells/MC for the initial inoculum, the higher was the percent of unoccupied MCs (with 1 cell/MC we had 35.6% of unoccupied MCs), which were shown to remain uncovered during the whole period of culture. With an initial inoculum of 4, 6 and 8 VERO cells/MC, respectively 46%, 76% and 83% of the MCs were totally covered by cells after 7 days, the cultures showing at this time, respectively, 5.1 2 10⁵, 8.8 2 10⁵ and 1.8 2 10⁶ cells/ml, which represented a biomass production of respectively 8.5x, 9.7x and 15.5x. When compared to 175 cm² T-flasks, using the same amount of medium, a VERO cell culture on 2 mg/ml of MCs offers about 10 times more available surface for cell growth and allowed the obtention of 7 times more cells. The optimization procedures concerning initial steps of VERO cell cultures, such as the attachment, spreading and growth as a function of parameters like initial cell inoculum and medium supplementation are of special interest mainly due to the perspective of a large use of VERO cell cultures for human viral vaccine production.     

Extracellular matrix controls myosin light chain phosphorylation and cell contractility through modulation of cell shape and cytoskeletal prestress

The mechanism by which vascular smooth muscle (VSM) cells modulate their contractility in response to structural cues from extracellular matrix remains poorly understood. When pulmonary VSM cells were cultured on increasing densities of immobilized fibronectin (FN), cell spreading, myosin light chain (MLC) phosphorylation, cytoskeletal prestress (isometric tension in the cell before vasoagonist stimulation), and the active contractile response to the vasoconstrictor endothelin-1 all increased in parallel. In contrast, MLC phosphorylation did not increase when suspended cells were allowed to bind FN-coated microbeads (4.5-microm diameter) or cultured on micrometer-sized (30 x 30 microm) FN islands surrounded by nonadhesive regions that support integrin binding but prevent cell spreading. Cell spreading and MLC phosphorylation also both decreased in parallel when the mechanical compliance of flexible FN substrates was raised. MLC phosphorylation was inhibited independently of cell shape when cytoskeletal prestress was dissipated using a myosin ATPase inhibitor in fully spread cells, whereas it increased to maximal levels when microtubules were disrupted using nocodazole in cells adherent to FN but not in suspended cells. These data demonstrate that changes in cell-extracellular matrix (ECM) interactions modulate smooth muscle cell contractility at the level of biochemical signal transduction and suggest that the mechanism underlying this regulation may involve physical interplay between ECM and the cytoskeleton, such that cell spreading and generation of cytoskeletal tension feed back to promote MLC phosphorylation and further increase tension generation.     

Structure-function correlation in airway smooth muscle adapted to different lengths

Airway smooth muscle is able to adapt and maintain a nearly constant maximal force generation over a large length range. This implies that a fixed filament lattice such as that found in striated muscle may not exist in this tissue and that plastic remodeling of its contractile and cytoskeletal filaments may be involved in the process of length adaptation that optimizes contractile filament overlap. Here, we show that isometric force produced by airway smooth muscle is independent of muscle length over a twofold length change; cell cross-sectional area was inversely proportional to cell length, implying that the cell volume was conserved at different lengths; shortening velocity and myosin filament density varied similarly to length change: increased by 69.4% ± 5.7 (SE) and 76.0% ± 9.8, respectively, for a 100% increase in cell length. Muscle power output, ATPase rate, and myosin filament density also have the same dependence on muscle cell length: increased by 35.4% ± 6.7, 34.6% ± 3.4, and 35.6% ± 10.6, respectively, for a 50% increase in cell length. The data can be explained by a model in which additional contractile units containing myosin filaments are formed and placed in series with existing contractile units when the muscle is adapted at a longer length. muscle contraction; myosin filaments; ATPase activity; electron microscopy     

Induction of spreading during fibroblast movement

This paper describes the phenomenon of retraction-induced spreading of embryonic chick heart fibroblasts moving in culture. Measurable criteria of cell spreading (increase in area of the spreading lamella, and total spread area of the cell) are found to change predictably with retraction of a portion of the cell margin. Ruffling activity was found to increase. The leading lamella of a spread fibroblast ordinarily advances slowly, with an average area increase of approximately 21 mu2m/min. A 10- to 30-fold increase in spreading occurs within 8 s after onset of retraction at the trailing edge and then decreases slightly so that by 1 min the increase in spreading is five to tenfold. During this period, there is a linear relationship between area increase at the leading edge and area decrease at the trailing edge. During the next 10--15 min, spreading gradually decreases to normal. Although the relationship between area spreading and area retracting of fibroblasts at different phases of movement is not significantly linear, it is highly correlated (Table II). These results suggest that the rate of fibroblast spreading may be inversely related to the degree of spreading of the cell as a whole.     

Modulation of contraction by gelation/solation in a reconstituted motile model

The actin-based cytoskeleton is a dynamic component of living cells with major structural and contractile properties involved in fundamental cellular processes. The action of actin-binding proteins can decrease or increase the gel structure. Changes in the actin-based cytoskeleton have long been thought to modulate the myosin II-based contractions involved in these cellular processes, but there has been some debate concerning whether maximal gelation increases or decreases contractile activity. To address this question, we have examined how contractile activity is modulated by the extent of actin gelation. The model system consists of physiologically relevant concentrations and molar ratios of actin filaments (whose lengths are controlled by gelsolin), the actin-cross-linking protein filamin, and smooth muscle myosin II. This system has been studied at the macroscopic and light microscopic levels to relate the gel structure to the rate of contraction. We present results which show that while a minimal amount of structure is necessary to transmit the contractile force, increasing the gel structure inhibits the rate of contraction, despite an increase in the actin-activated Mg(2+)-ATPase activity of myosin. Decreasing the total myosin concentration also inhibits the rate of contraction. Application of cytochalasin D to one side of the contractile network increases the rate of contraction and also induces movement comparable to flare streaming observed in isolated amoeba cytoplasm. These results are interpreted relative to current models of the relationship between the state of gelation and contraction and to the potential effects of such a relationship in the living cell.     

Effect of thyroid hormone on the contractility of self-organized heart muscle

Tissue-engineered heart muscle may provide an alternative treatment modality for end-stage congestive heart failure. We have previously described a method to engineer contractile heart muscle in vitro (termed cardioids). This study describes a method to improve the contractile properties of cardioids utilizing thyroid hormone (T3) stimulation. Cardioids were engineered by promoting the self-organization of primary neonatal cardiac cells into a contractile tissue construct. Cardioids were maintained in standard cell culture media supplemented with varying concentrations of T3 in the range 1-5ng/ml. The contractile properties of the cardioids were evaluated 48h after formation. Stimulation with T3 resulted in an increase in the specific force of cardioids from an average value of 0.52 +/- 0.16kPa (N = 6) for control cardioids to 2.42 +/- 0.29kPa (N = 6) for cardioids stimulated with 3ng/ml T3. In addition, there was also an increase in the rate of contraction and relaxation in response to T3 stimulation. Cardioids that were stimulation with T3 exhibited improved pacing characteristics in response to electrical pacing at 1-5Hz and an increase in the degree of spontaneous contractility. Changes in the gene expression of SERCA2, phospholamban, alpha-myosin heavy chain, and beta-myosin heavy chain correlated with the changes in contractile properties. This study demonstrates the modulation of the contractile properties of tissue-engineered heart muscle using T3 stimulation.     

Role of the Z band in the mechanical properties of the heart

In striated muscle the mechanism of contraction involves the cooperative movement of contractile and elastic components. This review emphasizes a structural approach that describes the cellular and extracellular components with known anatomical, biochemical, and physical properties that make them candidates for these contractile and elastic components. Classical models of contractile and elastic elements and their underlying assumptions are presented. Mechanical properties of cardiac and skeletal muscle are compared and contrasted and then related to ultrastructure. Information from these approaches leads to the conclusion that the Z band is essential for muscle contraction. Our review of Z band structure shows the Z band at the interface where extracellular components meet the cell surface. The Z band is also the interface from cell surface to myofibril, from extra-myofibrillar to myofibril, and finally from sarcomere to sarcomere. Our studies of Z band in defined physiologic states show that this lattice is an integral part of the contractile elements and can function as an elastic component. The Z band is a complex dynamic lattice uniquely suited to play several roles in muscle contraction.     

Changes in alpha-actinin localization and myofibrillogenesis in rat cardiomyocytes under cultivation

By indirect immunofluorescence with monoclonal anti-alpha-actinin antibodies the localization of this contractile protein was studied in ventricular cardiac myocytes from newborn 2-4-day old rats in the course of their cultivation. In freshly isolated heart muscle cells a predominant longitudinal orientation of myofibrils was observed; in some cells on the periphery of cytoplasm the contours of Z-lines are indistinct. During cell spreading, in the areas of intercalated discs, growing processes were observed mostly containing no contractile structures at earlier stages of cultivation. On days 3 to 14, the cytoplasmic processes and ruffles are filled with developing myofibrils. The cultures are heterogeneous in the morphology of contractile apparatus of individual cells. In most cardiomyocytes mature myofibrils are well-developed in the central part of the cytoplasm, whereas in its peripheral areas non-myofibrillar stress-fiber-like structures and bundles with continuous distribution of alpha-actinin frequently connected to myofibrils are more typical. In the areas of active myofibrillogenesis, located mainly on the cell periphery, numerous alpha-actinin dots are observed; most of them are arranged linearly and periodically at a distance of 0.3-1.5 microns and seem to be structural precursors of Z-lines. The data obtained show that the cultures of mammalian cardiac cells may be a convenient object for studying myofibrillogenesis in the course of cardiomyogenic differentiation.     

On the question of cyclic GMP as the mediator of the effects of acetylcholine on the heart

The effects of acetylcholine and sodium nitroprusside on cyclic GMP levels, contractile force, and glycogen metabolism were investigated in the perfused rat heart. While both agents produced time- and concentration-dependent increases in cyclic GMP, only acetylcholine significantly decreased contractile force. Neither agent altered the basal cyclic AMP concentration, cyclic AMP-dependent protein kinase activity ratio, or phosphorylase activity. When dosages were adjusted to give approximately equal increases in cyclic GMP, acetylcholine attenuated the effect of epinephrine on contractile force and glycogen phosphorylase activity while nitroprusside did not antagonize the action of the beta-adrenergic agent on either parameter. The data suggest that increased cardiac cyclic GMP is not sufficient to completely explain the action of acetylcholine on either contractile force or its antagonism of epinephrine-induced increases in force or glycogen phosphorylase activity.     

On the question of cyclic GMP as the mediator of the effects of acetylcholine on the heart.

The effects of acetylcholine and sodium nitroprusside on cyclic GMP levels, contractile force, and glycogen metabolism were investigated in the perfused rat heart. While both agents produced time- and concentration-dependent increases in cyclic GMP, only acetylcholine significantly decreased contractile force. Neither agent altered the basal cyclic AMP concentration, cyclic AMP-dependent protein kinase activity ratio, or phosphorylase activity. When dosages were adjusted to give approximately equal increases in cyclic GMP, acetylcholine attenuated the effect of epinephrine on contractile force and glycogen phosphorylase activity while nitroprusside did not antagonize the action of the beta-adrenergic agent on either parameter. The data suggest that increased cardiac cyclic GMP is not sufficient to completely explain the action of acetylcholine on either contractile force or its antagonism of epinephrine-induced increases in force or glycogen phosphorylase activity.     

Reduced vulnerability of the hypertrophied rat heart to oxygen-radical injury

Effects of xanthine--xanthine oxidase produced oxygen radicals were studied in hypertrophied rat hearts in a Langendorff preparation. Heart hypertrophy was produced by banding of the abdominal aorta for 6 weeks. This resulted in a 22% increase in ventricle/body weight ratio compared with that of sham-operated controls. Perfusion with xanthine--xanthine oxidase caused contractile failure and a significant rise in the resting tension. Complete contractile failure in hypertrophied hearts was seen at 25.5 +/- 3.2 min, whereas in control hearts it happened at 14.4 +/- 5.6 min. Contractile failure due to oxygen radicals in both groups was associated with a decline in high energy phosphates, increased lipid peroxidation, and extensive structural damage. Sarcolemma in both groups became permeable to the extracellular tracer lanthanum. As compared with control, in hypertrophied hearts the malondialdehyde content, indicative of lipid peroxidation, was less by 40%; whereas superoxide dismutase, a free radical scavenger, was higher by a similar amount. These data show a greater capacity of the 6-week hypertrophied heart to withstand a free radical induced contractile failure. This delay in oxygen radical effect can be partially explained by the reduced lipid peroxide content and increased superoxide dismutase activity in the hypertrophied hearts.     

Subcellular calcium transport during contractile failure and recovery in heart perfused with Na-free medium

Perfusion of isolated rat hearts with Na-free medium resulted in an immediate increase in contractile force followed by a decline and complete loss of contractile force within 25 s. The recovery of the contractile force upon reperfusion was only partial if the duration of Na-free perfusions was 10 min or longer. Ca binding and uptake activities of mitochondria obtained from hearts perfused with Na-free medium did not change significantly. However, Ca binding and uptake activities of microsomes were depressed after 5 min of perfusion. The critical concentration of Na in the perfusion medium for inducing these changes was found to be less than 35 mM. The microsomal Ca-ATPase activity was decreased after 10 min of Na-free perfusion. Only partial recovery of microsomal Ca uptake was observed upon reperfusion of hearts preperfused with Na-free medium for 20 min or longer whereas Ca-ATPase activity in these hearts did not recover at all. These results suggest that the defect in the microsomal Ca transport may be secondary to the development of contractile failure and may partially be associated with the inability of Na-depleted hearts to recover fully their contractile force.     

The effect of inoculum size on the growth of cell and root cultures ofHyoscyamus muticus: Implications for reactor inoculation

Cell suspensions inoculated at low cell concentrations displayed a typical growth reduction, whereas root cultures displayed an improvement in growth. Specific growth rate ofHyoscyamus muticus cell suspensions decreased from 0.25 to 0.12 d⁻¹ as inoculum concentration was reduced from 4.0 to 0.02 g fresh weight per liter. In contrast, roots show an increase in growth rate from 0.24 to 0.43 d⁻¹. These contrasting growth patterns can be explained as the result of: a) the high specific surface area of cells as compared to roots and, b) the differentiated structure of roots. The dispersed nature of cell suspensions makes them more prone to leakage of key growth factors/cellular contents to medium. The results of this work indicate that cell cultures require substantially higher inoculum concentrations. In contrast, roots can be inoculated at very low concentrations. These facts imply that whereas seed vessels must be employed by cell suspensions, their use for root cultures is a compromise between an easier handling of an entwined root mass and the reduction of the contamination risk of large medium volumes.     

Cryopreservation of heart cells from the eastern oyster

Summary Conditions were developed to cryopreserve cells from pronase-dissociated atria and ventricles of eastern oysters (Crassostrea virginica). The effect of three concentrations (5, 10, 15%) of the cryoprotectants (dimethyl sulfoxide, glycerol, and propylene glycol), three thawing temperatures (25, 45, 75°C), and three cooling rates (slow, medium, fast) were compared. Cells were frozen at −80°C and plunged in liquid nitrogen. Thawed cells were seeded in 96-well plates and primary cultures were evaluated after 3 d by measuring the metabolic activity using a tetrazolium compound, 3-(4,5-dimethylthiazol-2-yl)-5-( 3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, and by comparing the relative spreading of cells between treatments. The best conditions for freezing and thawing of cells for each cryoprotectant were selected and a final study was performed to compare cryoprotectants. For this final study, we measured the number of cells and their viability 3 d after thawing, in addition to determining cell metabolic activity and cell spreading. Primary cultures of cells frozen without cryoprotectant and of nonfrozen cells were used as controls in all studies. Atrial cells were best cryopreserved with glycerol at a concentration of 10%, a medium cooling rate, and thawing at 45°C. After thawing, atrial cells showed 53±5% of the metabolic activity, 84±5% of the number, and 92±2% of the viability of nonfrozen cells. For ventricular cells, 10% glycerol, a medium cooling rate, and thawing at 25°C yielded the best results. The thawed ventricular cells showed 83±5% of the metabolic activity, 91±5% of the number, and 96±2% of the viability of nonfrozen cells.     

Coordinate regulation of contractile protein synthesis during myoblast differentiation

The synthesis of contractile proteins has been studied during the differentiation of quail skeletal muscle myoblasts in culture. Myoblast differentiation was synchronized by transferring secondary cultures of rapidly dividing myoblasts into medium lacking cell division-promoting factors. Cultures at various stages of differentiation were then pulse-labeled with 35S-methionine, and cell extracts were resolved by electrophoresis on two-dimensional gels. Incorporation into specific proteins was quantitated by autoradiography and fluorography using a scanning densitometer. Contractile proteins synthesized by muscle cultures were identified by their co-electrophoresis on two-dimensional gels with contracile proteins purified from quail breast muscle. Our results show that the synthesis of myosin heavy chain, two myosin light chains, two subunits of troponin and two subunits of tropomyosin is first detected at the time of myoblast fusion and then rapidly increase at least 500 fold to maximum rates which remain constant in muscle fibers. Both the kinetics of activation and the molar rates of synthesis of these contractile proteins are virtually identical. Muscle-specific actin (alpha) synthesis also increases at the time of myoblast fusion, but this actin (alpha) is synthesized at 3 times the rate of other contractile proteins. The synthesis of 30 other muscle cell proteins was quantitated, and most of these are shown to follow different patterns of regulation. From these results, we conclude that the contractile proteins are regulated coordinately during myoblast differentiation.     

Contractile and Mechanical Properties of Epithelia with Perturbed Actomyosin Dynamics

Mechanics has an important role during morphogenesis, both in the generation of forces driving cell shape changes and in determining the effective material properties of cells and tissues. Drosophila dorsal closure has emerged as a reference model system for investigating the interplay between tissue mechanics and cellular activity. During dorsal closure, the amnioserosa generates one of the major forces that drive closure through the apical contraction of its constituent cells. We combined quantitation of live data, genetic and mechanical perturbation and cell biology, to investigate how mechanical properties and contraction rate emerge from cytoskeletal activity. We found that a decrease in Myosin phosphorylation induces a fluidization of amnioserosa cells which become more compliant. Conversely, an increase in Myosin phosphorylation and an increase in actin linear polymerization induce a solidification of cells. Contrary to expectation, these two perturbations have an opposite effect on the strain rate of cells during DC. While an increase in actin polymerization increases the contraction rate of amnioserosa cells, an increase in Myosin phosphorylation gives rise to cells that contract very slowly. The quantification of how the perturbation induced by laser ablation decays throughout the tissue revealed that the tissue in these two mutant backgrounds reacts very differently. We suggest that the differences in the strain rate of cells in situations where Myosin activity or actin polymerization is increased arise from changes in how the contractile forces are transmitted and coordinated across the tissue through ECadherin-mediated adhesion. Altogether, our results show that there is an optimal level of Myosin activity to generate efficient contraction and suggest that the architecture of the actin cytoskeleton and the dynamics of adhesion complexes are important parameters for the emergence of coordinated activity throughout the tissue.     

Cholinergic mechanisms of the hypothalamus medial preoptic area in control of thermoregulation and of wakefulness-sleep states in pigeons

The data obtained show that cholinergic mechanisms of the medial preoptic area of hypothalamus participate in control of wakefulness-sleep states and thermoregulation parameters in pigeons. Muscarinic and nicotinic cholinergic receptors are established to be involved in the wakefulness maintenance. The muscarinic cholinergic receptor activation of the medial preoptic area is accompanied by an elevation of the brain temperature, by development of peripheral vasoconstriction, and by an in increase in level of the muscle contractile activity. During the nicotinic cholinergic receptor activation of the area, a decrease in the brain temperature and an increase in level of the muscle contractile activity are found. A comparative analysis of experiments and early investigation suggests that during the cholinergic receptors activation changes in the brain temperature of pigeons depend on type of the cholinergic receptors but not on their localization in the preoptic area of hypothalamus.     

Mathematical model of cardiac mechanogram rhythmicity (based on mollusc heart)

1. The time course of force development by the heart is modelled by Gompertz kinetics from the product of two terms: a cumulative increase in relative number of activated "contractile units", and an exponential decrease in contractile force. 2. For each beat, an "initial condition" is specified by an "intrinsic tension" parameter, and a specific rate of change of tension; cardioactive agents change these specifications. 3. Depending on parameter values, heartbeats are predicted that are constant, or in which the frequency, amplitude and baseline tension are appropriate to inhibited or augmented cardiac activity.