Immunological quantitation and localization of tubulin in adult rat heart isolated myocytes

Isolated myocytes were purified from adult rat heart. Identification and localization of microtubules and quantitation of tubulin in these cells were performed by immunochemical procedures. Antibodies were raised against brain tubulin and purified by affinity chromatography. An enzyme-linked immunosorbent assay, ELISA, was developed for quantitation of tubulin. It allowed the measurement of 10 to 500 ng of tubulin. Tubulin content in adult rat cardiac myocytes was found to be approximately 10 micrograms per 100 mg of the total protein content. By means of a double immunofluorescence technique, the microtubule network, identified with antitubulin, was studied in reference to the sarcomeric A band labeled with antibodies specific to myosin heavy chains. The basis for identifying the microtubule network have included the use of specific antitubulin immunoglobulins and the sensitivity of the specific labeling of the network to antimitotic drugs and low temperature. It was found that microtubules were organized mainly around the nuclei, with important concentrations at the poles, showing extensions in the cone and in the cytoplasm as loosely organized loops. The shape of adult cardiac myocyte was not dependent upon the integrity of the microtubule network.     

Ouabain receptor in isolated adult dog heart myocytes

Ouabain binding was studied in isolated adult dog heart myocytes. The binding was correlated with the inhibition of K⁺-activated para-nitrophenylphosphatase (K⁺-PNPPase) activity and the beating response. It was shown that: (i) the specific binding was dependent upon Mg²⁺ and was inhibited by K⁺; (ii) the maximal binding capacity (B_max) was 7.4 × 10⁵ ouabain molecules per cell, or 410 pmol ouabain/K⁺-PNPPase unit (μmol/min); (iii) in the presence of Mg²⁺ (5 mm), there were two components in the Scatchard plot, i.e., a high-affinity component with a K_d value of 5.6 × 10^?8m and a low-affinity component with a K_d value of 6.7 × 10^?7m; (iv) the Hill coefficient (n′) for ouabain binding was 0.72 with a S_0.5 value of 7.1 × 10^?7m; these values were compatible with the values obtained from studies of K⁺-PNPPase inhibition by ouabain (n′ = 0.55, S_0.5 = 3.6 × 10^?7 m) and remained unchanged in the presence of physiological concentrations of Na⁺ plus K⁺; (v) in the presence of Mg²⁺ and K⁺, the high-affinity component tended to conform to the low-affinity component with an apparent decrease in B_max; (vi) in the presence of Mg²⁺ and para-nitrophenylphosphate, the low-affinity component was changed to the high-affinity component with no change in B_max; (vii) the dissociation rate of the labeled ouabain in the highly dilute medium was not altered in the presence of excess amounts of unlabeled ligand; this eliminated the possibility that the apparent negative cooperativity was due to a site-to-site interaction between receptors; (viii) ouabain increased the number of beating cells and the frequency of beating. Based on these findings, it is concluded that: (i) isolated myocytes possess functional receptors for ouabain; (ii) the binding of ouabain is associated with its inhibition of K⁺-PNPPase activity; (iii) ouabain receptors in isolated myocytes are of one class with at least two interconvertible conformational states.     

Tolerance to physiological calcium by isolated myocytes from the adult rat heart; An improved cellular preparation

The preparation of isolated adult rat heart myocytes able to tolerate physiological calcium concentrations is described. The use of tissue culture medium as the buffer for the enzymic perfusion and digestion of the heart and, subsequently, not exposing isolated myocytes to temperatures below room temperature, proved necessary for the preparation of isolated myocytes able to maintain integrity in the presence of 2 mM calcium. After 60 minutes, 85 percent of the myocytes incubated at 37° with 2 mM calcium exclude the dye trypan blue. Levels of ATP and related compounds, although depressed, were maintained for an extended period. Oxidation of glucose and pyruvate was greater and succinate oxidation lower in calcium-resistant mycoytes compared to myocytes not resistant to calcium. The two types of myocytes were equally rapid in converting succinate to malate. Oxygen utilization increased following the addition of calcium. Myocytes demonstrated both Pasteur and Randle effects and responded to plasma levels of insulin by increasing glucose oxidation. This is the first report of isolated adult rat heart myocytes able to tolerate millimolar calcium concentrations in physiologic medium.     

Immunofluorescence and immunogold electron microscopy of desmin in isolated adult heart myocytes of the rat

Isolated myocytes of the adult mammalian heart are useful for studying cytoskeletal changes during development of irreversible myocardial injuries. Using monoclonal antibodies we have studied the structural organization of desmin in freshly isolated cardiomyocytes from rat hearts. This preparation consists of approximately 85% calcium tolerant rod shaped cells and 15% contracted "square cells" and "round cells" that were initially injured during separation. Cells were quick-frozen at -196 degrees C without any chemical stabilization, cryosectioned and then further processed for immunofluorescence or immunoelectron microscopy. Freshly isolated rod shaped cells exhibit the specific pattern of interfibrillar desmin organization of striated muscle. Furthermore, high resolution immunogold preparations show that desmin in the rod cells occurs in apposition to the edges of the Z-bands as well as closely associated with the plasmalemma. We could find no evidence for the presence of desmin within the Z-band plaques. This organization of desmin is completely absent in the contracted round cells. Thus, already at advanced stages of square cell development, desmin is almost entirely confined to the outer areas of the central filamentous core. We conclude that during the process of square cell contracture, the filamentous desmin contacts with Z-bands and sarcolemma are broken, leading to the unorganized array of desmin in round cells.     

Immunofluorescence and immunogold electron microscopy of desmin in isolated adult heart myocytes of the rat

Summary Isolated myocytes of the adult mammalian heart are useful for studying cytoskeletal changes during development of irreversible myocardial injuries. Using monoclonal antibodies we have studied the structural organization of desmin in freshly isolated cardiomyocytes from rat hearts. This preparation consists of approximately 85% calcium tolerant rod shaped cells and 15% contracted square cells and round cells that were initially injured during separation. Cells were quick-frozen at –196° C without any chemical stabilization, cryosectioned and then further processed for immunofluorescence or immunoelectron microscopy. Freshly isolated rod shaped cells exhibit the specific pattern of interfibrillar desmin organization of striated musele. Furthermore, high resolution immunogold preparations show that desmin in the rod cells occurs in apposition to the edges of the Z-bands as well as closely associated with the plasmalemma. We could find no evidence for the presence of desmin within the Z-band plaques. This organization of desmin is completely absent in the contracted round cells. Thus, already at advanced stages of square cell development, desmin is almost entirely confined to the outer areas of the central filamentous core. We conclude that during the process of square cell contracture, the filamentous desmin contacts with Z-bands and sarcolemma are broken, leading to the unorganized array of desmin in round cells.     

Effects of amiloride on calcium uptake by myocytes isolated from adult rat hearts

Amiloride at high concentrations inhibits the uptake of Ca by rat heart myocytes containing elevated levels of intracellular Na and retards the development of Ca-dependent hypercontracture in these cells. In contrast, amiloride enhances the net uptake of Ca in Ca-tolerant myocytes containing normal levels of Na. The results suggest that amiloride may inhibit Na-Ca exchange across the sarcolemma of cardiac myocytes.     

Glucose and octanoate utilization by isolated adult rat heart cells

Rat heart muscle cells continue to beat in the isolated state apparently independent of any innervation. The oxidation of ¹⁴C-glucose to ¹⁴CO₂ was linear for at least 60 minutes of incubation. The rate of glucose oxidation rose rapidly up to a medium glucose concentration of 2.5 mM and then plateaued. Lactate production reached a maximum at 5 mM glucose. Glucose uptake was linearly related to the concentration up to 40 mM. The addition of octanoate reduced, but did not eliminate, glucose oxidation. Octanoate utilization increased with increasing concentration and reached a maximum at 2 mM. The oxidation of octanoate was linearly related to the time of incubation for at least 90 minutes. The presence of glucose, at a concentration of 1.25 mM or higher, increase the oxidation of octanoate by the heart cells. The metabolic parameters measured with the isolated heart cells gave values comparable to those obtained with the perfused rat heart. Decreasing or increasing the concentration of sodium, potassium or magnesium did not effect the oxidation of either glucose or octanoate with the exception that when sodium was increased above 200 mM, a significant increase in glucose oxidation was observed. In contrast, the addition of calcium to a calcium free medium increased glucose oxidation, reaching a maximum at 0.2 mM calcium. The oxidation of octanoate reached a maximum at 0.2 mM and then decreased significantly with increasing calcium concentration. The metabolic activity appears to be independent of the concentration of sodium, potassium or magnesium. In contrast, the isolated heart cell is very sensitive to a change in calcium concentration.     

Metabolism and salvage of adenine and hypoxanthine by myocytes isolated from mature rat heart

Adenine and hypoxanthine can be utilised by cardiac muscle cells as substrates for the synthesis of ATP. A possible therapeutic advantage of these compounds as high-energy precursors is their lack of vasoactive properties. Myocytes isolated from mature rat heart have been used to establish in kinetic detail the capacity of the heart to incorporate adenine, hypoxanthine and ribose into cellular nucleotides. Maximum rates of catalysis by enzymes on the salvage pathways have been established. Whilst the rate of incorporation of adenine into the ATP pool appears to depend upon intracellular concentrations of adenine and phosphoribosylpyrophosphate, for hypoxanthine the pattern is more complex. Hypoxanthine is salvaged at a slow rate compared with adenine, and is incorporated into GTP and IMP as well as into adenine nucleotides. The rate of incorporation of hypoxanthine into both IMP and ATP is accelerated in myocytes incubated with ribose. However, the rate-limiting reaction appears to be that catalysed by adenylosuccinate synthetase, for the rate of ATP synthesis is not accelerated when hypoxanthine concentration is increased from 10 to 50 microM, while the rate of IMP synthesis is more than doubled. Adenine and hypoxanthine phosphoribosyl transferases are present in equal catalytic amounts, but rat cardiac myocytes have very little adenylosuccinate synthetase activity. Exogenous ribose is incorporated into adenine nucleotides in amounts equimolar with adenine or hypoxanthine.     

Degradation and resynthesis of adenine nucleotides in adult rat heart myocytes

The degradation and short-term resynthesis of adenine nucleotides have been examined in a preparation of isolated rat heart myocytes. These myocyte preparations are essentially free of vascular and endothelial cells, contain levels of adenine nucleotides quite comparable to those of intact heart tissue, and retain these components remarkably well for up to 2 h of aerobic incubation in the presence of 1 mM Ca2+. When the cells are rapidly and synchronously de-energized by addition of uncoupler, an inhibitor of respiration and iodoacetate, cellular ATP is degraded almost quantitatively to AMP. The AMP is then converted to either intracellular adenosine, which accumulates to high concentrations before release to the cell exterior, or to IMP. The relative contribution of these two pathways depends on the metabolic state of the cells just prior to de-energization, with IMP production favored when respiring cells are de-energized and adenosine formation predominant when glycolyzing myocytes are subjected to this treatment. Cells de-energized by anaerobiosis in the absence of glucose lose ATP and adenine nucleotides with the production of IMP and adenosine. Upon reoxygenation, these cells restore a high adenylate energy charge and about 60% of control levels of GTP. There is a net resynthesis of 5-7 nmol of adenine nucleotides.mg-1 protein with a corresponding decline in IMP. Added [14C]adenosine labels the adenine nucleotide pool, but little net resynthesis of adenine nucleotides via adenosine kinase can be detected. It therefore appears that a rapid regeneration of adenine nucleotides can occur via the enzymes of the purine nucleotide cycle in heart myocytes and is limited by the size of the IMP pool retained.     

Isolation of Ca2+-tolerant myocytes from adult rat heart

A procedure for the isolation of myocytes from adult rat hearts is described. It is based on successive treatments with Ca2+-free medium, disaggregating enzymes (collagenase and hyaluronidase) and mechanical agitation. Several recent isolation methods were compared and their best features were combined, together with some original modifications. A good yield of high purity myocytes with excellent morphological and functional integrity was obtained. The cells are tolerant to physiological concentrations of Ca2+. Cellular levels of ATP, Na+, and K+ are close to those in intact hearts and glucose oxidation rates and succinate exclusion are also close to normal. These characteristics are maintained for periods over 1 h.     

Oxidation of myoglobin in isolated adult rat cardiac myocytes by 15-hydroperoxy-5,8,11,13-eicosatetraenoic acid

The oxidation of intracellular myoglobin by 15-hydroperoxy-5,8,11,13-eicosatetraenoic acid was studied in suspensions of isolated adult rat heart cells. Myoglobin was converted to a species identified as ferrylMb by its reaction with Na2S to form ferrous sulfmyoglobin. This process was time-dependent and concentration-dependent in a manner consistent with direct accessibility of the exogenous peroxide to the cytosolic protein. The results indicate that myoglobin oxidation may be an early sign of oxidative injury and may limit myocardial function by elimination of this short-term O2 reserve.     

Effects of insulin on glucose metabolism in isolated heart myocytes from adult rats

The study examined the effect of insulin on glucose metabolism in freshly isolated calcium-tolerant heart myocytes from adult rats. The uptake of 2-deoxyglucose demonstrated an initial lag in response to insulin and the maximal insulin effect was not attained until after 3 min preincubation with the hormone. A dose-response study of 14CO2 production from [14C]glucose revealed that the maximum insulin stimulation of glucose utilization occurred with 5 mU/ml. Both the uptake and the oxidation of glucose proceeded at a linear rate in the absence and presence of insulin. However, insulin exerted a greater effect on the uptake (42-54%) than on the oxidation (17-22%) of exogenous glucose. Incorporation of glucose into glycogen was markedly increased by insulin and resulted in the myocyte glycogen concentration returning to in vivo levels. In the absence of insulin, glucose incorporation plateaued within 10 min of incubation and the glycogen concentration was not altered. Our findings also indicate that at equilibrium, insulin-treated cells exhibited a higher glycogen turnover rate. It thus appears that insulin exerts a differential effect on the different pathways in glucose metabolism in the isolated cardiac cells. This may be related in part to their quiescent state and lower energy demand.     

Structural and functional properties of adult rat heart myocytes lysed with digitonin

Low concentrations of digitonin disrupt the sarcolemma of adult rat heart myocytes selectively and completely. When the digitonin lysis is carried out in the presence of 10 mM Mg-ATP, the permeabilized cells retain the rod-cell morphology typical of heart cells in situ and show spontaneous phasic contractions. The rate of contraction is a function of the free Ca2+ concentration from a pCa of 7.2 to 5.2. Higher levels of free Ca2+ result in hypercontracture of the myocytes into round cells with characteristically distorted morphology. The sarcoplasmic reticulum of digitonin-lysed myocytes takes up Ca2+ in an ATP-dependent reaction that is inhibited and reversed by caffeine and strongly enhanced by procaine or ruthenium red. The Ca2+ accumulation has a Km of 0.6 microM Ca2+, depends on Pi (Km of 13 mM), and is strongly inhibited by bicarbonate ion. The hypercontracture of digitonin-lysed myocytes is a function of both the pCa and the Mg-ATP concentration of the suspending medium. Hypercontracture requires ATP. Hypercontracture due to Ca2+ overload occurs at lower Ca2+ concentrations when Mg-ATP is decreased from 10 to 1 mM. However, at low concentrations of Mg-ATP (in the range from 1 to 10 microM), hypercontracture also occurs and is essentially Ca2+-independent. Since hypercontracture of heart myocytes appears analogous to the formation of contraction bands in situ, these observations may be relevant to the phenomena of oxygen paradox and of Ca2+ paradox in intact myocardial tissue.     

Associations between beta-tubulin and mitochondria in adult isolated heart myocytes as shown by immunofluorescence and immunoelectron microscopy

We have investigated the associations between beta-tubulin and mitochondria in freshly isolated cardiac myocytes from the rat. Beta-tubulin was identified by using monoclonal antibodies for immunofluorescence and high resolution immunogold electron microscopy. In addition, conventional transmission and scanning electron microscopic studies were performed. After chemical stabilization in a formaldehyde solution, the myocytes were shock-frozen at -150 degrees C, cryosectioned at -70 degrees C and subsequently processed for immunohistochemical and immunocytochemical microscopy. A characteristic of the rod shaped myocytes is the presence of a dense network of microtubules in the cytoplasm displaying a pattern of strong anti-beta-tubulin reaction. The complexity of this network however varies considerably among the myocytes reflecting microtubule dynamic instability. Further, our findings demonstrate that the beta-tubulin label in rod cells is confined to the perinuclear and interfibrillar spaces and, therefore, is largely colocalized with the cytoplasmic organelles. In myocytes undergoing severe contracture the distribution of beta-tubulin is entirely restricted to the outer mitochondrial-containing domain. This implies that, in a cell model with marked segregation of the contractile filaments and organelles, mitochondria are codistributed with microtubules in the total absence of desmin intermediate filaments. Moreover, our immunogold preparations demonstrate anti-beta-tubulin labelling in the outer mitochondrial membrane as well as of fibres in close apposition to this membrane. These results indicate the presence of a specific beta-tubulin binding to the outer mitochondrial membrane that probably also involves microtubule based translocators and/or MAPs.     

Associations between beta-tubulin and mitochondria in adult isolated heart myocytes as shown by immunofluorescence and immunoelectron microscopy

Summary We have investigated the associations between -tubulin and mitochondria in freshly isolated cardiac myocytes from the rat. Beta-tubulin was identified by using monoclonal antibodies for immunofluorescence and high resolution immunogold electron microscopy. In addition, conventional transmission and scanning electron microscopic studies were performed. After chemical stabilization in a formaldehyde solution, the myocytes were shock-frozen at –150°C, cryosectioned at –70°C and subsequently processed for immunohistochemical and immunocytochemical microscopy. A characteristic of the rod shaped myocytes is the presence of a dense network of microtubules in the cytoplasm displaying a pattern of strong anti--tubulin reaction. The complexity of this network however varies considerably among the myocytes reflecting microtubule dynamic instability. Further, our findings demonstrate that the -tubulin label in rod cells is confined to the perinuclear and interfibrillar spaces and, therefore, is largely colocalized with the cytoplasmic organelles. In myocytes undergoing severe contracture the distribution of -tubulin is entirely restricted to the outer mitochondrial-containing domain. This implies that, in a cell model with marked segregation of the contractile filaments and organelles, mitochondria are codistributed with microtubules in the total absence of desmin intermediate filaments. Moreover, our immunogold preparations demonstrate anti--tubulin labelling in the outer mitochondrial membrane as well as of fibres in close apposition to this membrane. These results indicate the presence of a specific -tubulin binding to the outer mitochondrial membrane that probably also involves microtubule based translocators and/or MAPs.     

Intracellular free Ca2+ and the hypercontracture of adult rat heart myocytes

The Ca2+ sensitivity of a population of isolated adult rat heart myocytes has been related to the Na+ content of the cells prior to Ca2+ exposure, and the intracellular free Ca2+ as reported by quin2 fluorescence when the cells are challenged with millimolar external Ca2+. Myocytes exposed to Ca2+ during quin2 loading show a resting intracellular free Ca2+ of 150 +/- 30 nM and retain the rod cell morphology of heart cells in situ. The myocytes take up Na+ and lose K+ when incubated in the cold in the absence of Ca2+. Large numbers of these rod-shaped, Na+-loaded myocytes hypercontract into grossly distorted round cell forms when exposed to physiological levels of Ca2+. The number of cells that hypercontract is proportional to the Na+ content of the cells prior to Ca2+ addition and can be directly related to the intracellular free Ca2+ concentration attained following Ca2+ addition. Fifty percent of the cells in a myocyte population hypercontract when the internal free Ca2+ concentration reported by quin2 reaches 400 nM and virtually all of the cells hypercontract when this value reaches 1 microM. The entry of Ca2+ into Na+-loaded myocytes is biphasic with one phase inhibited by Ca2+ channel blockade. This suggests that Ca2+ enters Na+-loaded myocytes by the Ca2+ channel as well as by Na+/Ca2+ exchange.     

Modulation of uncoupler-induced sugar uptake in isolated adult rat heart cells by isoproterenol

When cells (2.4 mg/ml) in the presence of glucose were exposed to 0.15 microM p-trifluoromethoxyphenylhydrazone (FCCP), the time until 50% of the rod-shaped cells had undergone contracture was more than twice as long for cells without isoproterenol as for cells with isoproterenol. The cause of this large effect was revealed in experiments without glucose where 3-O-methylglucose entry, ATP levels, and cellular configuration were measured simultaneously. It was found that the onset of contracture was almost coincident with the decline in total measured ATP, suggesting that, in any cell, contracture was accompanied by a sudden and total ATP loss. In control cells, FCCP stimulated 3-O-methylglucose entry at or before the time this ATP catastrophe occurred. In cells exposed to isoproterenol, however, the stimulation of 3-O-methylglucose entry by FCCP did not occur until after the ATP catastrophe, and the extent of stimulation was reduced. This suggests that, when glucose was present, the FCCP-induced glucose influx was sufficient to significantly delay the onset of contracture in control cells but not in cells treated with isoproterenol. This conclusion was borne out by the observation that the effect of isoproterenol on contracture could be overcome with insulin.