Characterization of contractions in enzymatically isolated rat ventricular myocytes: effects of ouabain and rubidium

The role of sarcolemma and especially sodium pump activity in the control of phasic contractile activity of Ca2+ tolerant myocytes was studied using ouabain and rubidium as sodium pump inhibitors. Initially, ouabain increased both the amplitude of shortening and the frequency of phasic contractions. Later, the amplitude began to decline whereas the frequency of beating continued to rise, often terminating in a steady contracture of the myocyte. Rubidium caused a rapid rise of beating frequency, which reached its full effect within 1-5 min and remained steady after that. The stimulation of contraction frequency and the inhibition of Na+-K+ ATPase were correlated in the case of ouabain but not in the case of rubidium. The results suggest that the stimulation of phasic contractions may be caused by increased uptake of cellular calcium through Na+-Ca+ exchange as a consequence of sodium pump inhibition and (or) depolarization of the sarcolemma by ouabain and rubidium.     

Role of myoglobin in the oxygen supply to red skeletal muscle.

The contribution of nyoglobin to the oxygen uptake of red skeletal muscle was estimated from the difference in oxygen uptake with and without functional myoglobin. The oxygen uptake of bundles (25 mm long, 0.5 mm mean diameter) of muscle fibers teased from pigeon breast muscle was measured in families of steady states of oxygen pressure from 0 to 250 mm Hg. The oxygen-binding function of myoglobin, in situ in muscle fiber bundles, was abolished by treatment with nitrite of hydroxylamine, which convert oxymyoglobin in situ to high spin ferric myoglobin, or with phenylhydrazine, which converts oxymyoglobin to denatured products, or with 2-hydroxyethylhydrazine, which appears to remove myoglobin from the muslce. The oxygen uptake was again measured. At higher oxygen pressure, where oxygen availability does not limit the respiration of the fiber bundles, oxygen uptake is not affected by any of the four reagents, which is evidence that mitochondrial oxygen uptake is not impaired. At lower oxygen pressure, where oxygen uptake is one-half maximal, the steady state oxygen consumption is roughly halved by abolishing functional myoglobin. Under the steady state conditions studied, the storage function of myoglobin, being static, vanishes and the transport function stands revealed. We conclude from these experiments that myoglobin may transport a significant fraction of the oxygen consumed by muscle mitochondria.     

Nicotinamide adenine dinucleotide fluorescence spectroscopy and imaging of isolated cardiac myocytes.

Nicotinamide adenine dinucleotide (NADH) plays a critical role in oxidative phosphorylation as the primary source of reducing equivalents to the respiratory chain. Using a modified fluorescence microscope, we have obtained spectra and images of the blue autofluorescence from single rat cardiac myocytes. The optical setup permitted rapid acquisition of fluorescence emission spectra (390-595 nm) or intensified digital video images of individual myocytes. The spectra showed a broad fluorescence centered at 447 +/- 0.2 nm, consistent with mitochondrial NADH. Addition of cyanide resulted in a 100 +/- 10% increase in fluorescence, while the uncoupler FCCP resulted in a 82 +/- 4% decrease. These two transitions were consistent with mitochondrial NADH and implied that the myocytes were 44 +/- 6% reduced under the resting control conditions. Intracellular fluorescent structures were observed that correlated with the distribution of a mitochondrial selective fluorescent probe (DASPMI), the mitochondrial distribution seen in published electron micrographs, and a metabolic digital subtraction image of the cyanide fluorescence transition. These data are consistent with the notion that the blue autofluorescence of rat cardiac myocytes originates from mitochondrial NADH.     

Positive inotropic effects of ouabain in isolated cat ventricular myocytes in sodium-free conditions

The inotropic and toxic effects of cardiac steroids are thought to result from Na(+)-K(+)-ATPase inhibition, with elevated intracellular Na(+)(Na)causing increased intracellular Ca(2+)(Ca) via Na-Ca exchange. We studied the effects of ouabain on cat ventricular myocytes in Na(+)-free conditions where the exchanger is inhibited. Cell shortening and Ca transients (with fluo 4-AM fluorescence) were measured under voltage clamp during exposure to Na(+)-free solutions [LiCl or N-methyl-D-glucamine (NMDG) replacement]. Ouabain enhanced contractility by 121 +/- 55% at 1 micromol/l (n = 11) and 476 +/- 159% at 3 micromol/l (n = 8) (means +/- SE). Ca transient amplitude was also increased. The inotropic effects of ouabain were retained even after pretreatment with saxitoxin (5 micromol/l) or changing the holding potential to -40 mV (to inactivate Na(+) current). Similar results were obtained with both Li(+) and NMDG replacement and in the absence of external K(+), indicating that ouabain produced positive inotropy in the absence of functional Na-Ca exchange and Na(+)-K(+)-ATPase activity. In contrast, ouabain had no inotropic response in rat ventricular myocytes (10-100 micromol/l). Finally, ouabain reversibly increased Ca(2+) overload toxicity by accelerating the rate of spontaneous aftercontractions (n = 13). These results suggest that the cellular effects of ouabain on the heart may include actions independent of Na(+)-K(+)-ATPase inhibition, Na-Ca exchange, and changes in Na.     

Cytoplasmic free calcium measured by quin2 fluorescence in isolated ventricular myocytes at rest and during potassium-depolarization

We have measured intracellular free Ca2+ concentration in rat and guinea pig ventricular myocytes using the fluorescent Ca2+-indicator quin2. Our results indicate a resting concentration in heart cells that is considerably lower than previous estimates. The mean value of 137 nM for rat ventricle that we have recorded is consistent with the hypothesis that resting intracellular [Ca2+] is controlled by a voltage-dependent, sarcolemmal exchange mechanism. Furthermore, we show that activation of plasma membrane Ca2+-channels is involved when intracellular free Ca2+ increases in response to K+-depolarization.     

Monoamine oxidase, an intracellular probe of oxygen pressure in isolated cardiac myocytes

The activity of monamine oxidase, an enzyme located almost exclusively at the outer mitochondrial membrane, toward the substrate phenylethylamine is used to report the oxygen pressure at the outer mitochondrial membrane of intact cardiac myocytes isolated from hearts of adult rats. The rate of substrate oxidation, under the conditions used, follows the Michaelis-Menten relation, and accordingly can be used as a measure of the local chemical activity of dissolved oxygen. The oxygen pressure at the outer mitochondrial membrane of myocytes, at rest and after 2- to 3-fold stimulation of respiratory oxygen consumption, differs from the extracellular oxygen pressure by at most 2 torr. This implies that most of the large, about 20 torr, difference in oxygen pressure between capillary lumen and mitochondria of the working heart must be extracellular. At physiologically relevant concentrations of the substrates phenylethylamine and norepinephrine, monoamine oxidase activity is relatively insensitive to extracellular oxygen pressure in the range 155 to 8 torr, suggesting a limited role for regulation of biogenic amine oxidation by oxygen availability.     

The effects of low concentrations of caffeine on spontaneous Ca release in isolated rat ventricular myocytes

We have investigated the effects on spontaneous SR Ca release of modulating the sarcoplasmic reticulum ryanodine receptor (RyR) with low (<0.5 mM) concentrations of caffeine. Experiments were performed on isolated rat ventricular myocytes. Intracellular Ca concentration was measured with Indo-1 or Fluo-3 in voltage-clamped cells. Spontaneous Ca release was produced by elevating external Ca to 5 mM. Caffeine application increased the frequency of spontaneous release. Both the magnitude of the spontaneous Ca transients and the integral of the resulting Na-Ca exchange current were decreased by caffeine. The combination of increased frequency of spontaneous release and decreased Ca efflux per event meant that the Ca efflux per unit time was unaffected by low concentrations of caffeine. The SR Ca content was reduced by caffeine. The extra Ca efflux calculated from the Na-Ca exchange current integrals occurring during the initial burst of spontaneous activity on application of caffeine accounted for this reduction of SR Ca content. In contrast to these maintained effects on spontaneous release, caffeine had only transient effects on stimulated Ca release produced by depolarizing pulses. We conclude that stimulation of the RyR results in spontaneous release at SR Ca contents lower than those at which release would normally occur. Therefore, the balance between normal and spontaneous Ca release can be shifted by modulation of the RyR. This will have important consequences for arrhythmogenesis due to spontaneous Ca release.     

kappa-Opioid receptor stimulation increases intracellular free calcium in isolated rat ventricular myocytes.

The effect of two specific kappa-agonists, dynorphinA1-13 and U50,488H, on intracellular free calcium [Ca]i in isolated rat ventricular myocytes was studied. A spectrofluorimetric method using fura 2 as calcium indicator was employed. It was found that both agonists increased [Ca]i dose-dependently. The effect was attenuated by Mr 2266, a kappa-antagonist, indicating that the effect is a kappa-receptor mediated event. The effect was abolished by pretreatment with ryanodine, a drug that mobilizes calcium from the sarcoplasmic reticulum. It was, however, not affected by nifedipine, a calcium antagonist or removal of external calcium. The results indicate that the increase in [Ca]i due to kappa-opioid receptor stimulation results primarily from mobilization of calcium from an intracellular pool.     

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.     

Properties of L-type calcium channel gating current in isolated guinea pig ventricular myocytes.

Nonlinear capacitative current (charge movement) was compared to the Ca current (ICa) in single guinea pig ventricular myocytes. It was concluded that the charge movement seen with depolarizing test steps from -50 mV is dominated by L-type Ca channel gating current, because of the following observations. (a) Ca channel inactivation and the immobilization of the gating current had similar voltage and time dependencies. The degree of channel inactivation was directly proportional to the amount of charge immobilization, unlike what has been reported for Na channels. (b) The degree of Ca channel activation was closely correlated with the amount of charge moved at all test potentials between -40 and +60 mV. (c) D600 was found to reduce the gating current in a voltage- and use-dependent manner. D600 was also found to induce "extra" charge movement at negative potentials. (d) Nitrendipine reduced the gating current in a voltage-dependent manner (KD = 200 nM at -40 mV). However, nitrendipine did not increase charge movement at negative test potentials. Although contamination of the Ca channel gating current from other sources cannot be fully excluded, it was not evident in the data and would appear to be small. However, it was noted that the amount of Ca channel gating charge was quite large compared with the magnitude of the Ca current. Indeed, the gating current was found to be a significant contaminant (19 +/- 7%) of the Ca tail currents in these cells. In addition, it was found that Ca channel rundown did not diminish the gating current. These results suggest that Ca channels can be "inactivated" by means that do not affect the voltage sensor.     

Excitation-contraction coupling in isolated adult ventricular myocytes from the rat, dog, and rabbit: effects of various inotropic interventions in the presence of ryanodine

Enzymatically isolated ventricular cells from rats, dogs, and rabbits were electrically stimulated and their membrane potentials were recorded simultaneously with their contractions. Specific pharmacological interventions were used to assess the relative roles of transsarcolemmal Ca2+ entry and the Ca2+ release by the sarcoplasmic reticulum in activating contractions, in these myocytes. We used ryanodine and caffeine to influence Ca2+ release by the sarcoplasmic reticulum, BAY K 8644 and epinephrine to increase Ca2+ entry through Ca2+ channels, and veratridine, ouabain, and monensin to increase Ca2+ entry through Na+-Ca2+ exchange. Ryanodine (1 microM) completely inhibited the shortenings in rat and dog myocytes, but the contractions in rabbit myocytes were much less sensitive to this alkaloid. Similar inhibitory effects of ryanodine were observed in the presence of various inotropic agents with two exceptions: caffeine's effect on the dog myocytes was relatively insensitive to ryanodine and the long-lasting tonic contractions that veratridine triggered in the myocytes of all three species remained completely unaffected by ryanodine. The data indicate that contractile activation in rat and dog ventricular cells is strongly dependent on Ca2+ release from the sarcoplasmic reticulum, while contractility in rabbit myocytes seems to be more dependent on Ca2+ entry through the sarcolemma. The ryanodine-resistant tonic contractions triggered in the myocytes of all three species in the presence of veratridine may be activated by an increased Ca2+ entry via Na+-Ca2+ exchange.     

Properties of ventricular myocytes isolated from the hypertrophied and failing hearts of spontaneously hypertensive rats.

OBJECTIVE: To investigate how the morphological and physiological properties of single myocytes isolated from the hypertrophied, failing left ventricles (LV) differ from those of normal or hypertrophied not failing ventricles. METHOD: Single myocytes were isolated separately from right (RV) and left ventricles (LV) of male spontaneously hypertensive rats (SHR) or Wistar-Kyoto (WKY) rats at the age of 6 and 12 months and of SHRs which developed or not developed heart failure at the age of 20-24 months. We measured cells dimensions, range and kinetics of electrically stimulated or initiated by caffeine contractions and Ca2+ transients, and investigated the response of cells to thapsigargin. RESULTS: The transversal dimensions of the LV myocytes of 6 months old SHRs showed approximately 20% increase with respect to transversal dimensions of their RV myocytes and LV and RV myocytes of WKY rats. The difference did not change with progressing age and in the heart failure. The LV myocytes of 6 or 12 months old SHRs showed slowed kinetics of the Ca2+ transients and of contraction and relaxation and decreased contractile response to 2 s superfusion with 15 mM caffeine preceded by 5 mM Ni2+ used as an index of the sarcoplasmic reticulum (SR) Ca2+ content. Despite of this the range of shortening and relative contribution of the SR to contraction (assessed by measuring of the residual contractile response to electrical stimulation in cells poisoned with thapsigargin) or relaxation (assessed by calculation of the ratio of rate constants of the electrically stimulated and stimulated by 30 s superfusion with caffeine Ca2+ transients) was not altered in the hypertrophied myocytes. Properties of the LV myocytes of the 20-24 old SHRs with or without heart failure did not differ from those of LV myocytes of younger SHRs. The contractile response to caffeine of their RV myocytes dropped to the level of that in the LV myocytes. CONCLUSION: Our results suggest that transition from the compensated hypertrophy to the heart failure in 20-24 months old SHRs did not result from the further changes in properties of the surviving myocytes. Data from literature suggest that myocyte apoptosis and remodeling of the extramyocyte space is the more likely reason.     

Adverse effects of atrioventricular synchronous right ventricular pacing on left ventricular sympathetic activity, efficiency, and hemodynamic status

Right ventricular (RV) pacing is now recognized to play a role in the development of heart failure in patients with and without underlying left ventricular (LV) dysfunction. We used the cardiac norepinephrine spillover method to test the hypothesis that RV pacing is associated with cardiac sympathetic activation. We studied 8 patients with normal LV function using temporary right atrial and ventricular pacing wires. All measurements were carried out during a fixed atrial pacing rate. The radiotracer norepinephrine spillover technique was employed to measure total body and cardiac sympathetic activity while changes in LV performance were evaluated with a high-fidelity manometer catheter. Atrioventricular synchronous RV pacing, compared with atrial pacing alone, was associated with a 65% increase in cardiac norepinephrine spillover, an increase in LV end-diastolic pressure, and a reduction in myocardial efficiency. These responses may play a role in the development of heart failure and poor outcomes that are associated with chronic RV pacing.     

NADH fluorescence and oxygen uptake responses of hybridoma cultures to substrate pulse and step changes

A fiber-optic probe was interfaced to an analytical spectrofluorophotometeru and used to measure NAD(P)H fluorescence of hybridoma cells in a bioreactor. NAD(P)H fluorescence was found to qualitatively represent metabolic state during various induced metabolic transitions. NAD(P)H fluorescence increased immediately following aerobic-anaerobic transitions, and decreased immediately upon transition back to aerobic metabolism. Pulsing of glucose to glucose-depleted cultures caused NAD(P)H fluorescence to first increase immediately after the pulse, and then decrease gradually before reaching a constant level. Pulsing of glutamine to glutamine-depleted cultures resulted in a gradual increase in NAD(P)H fluorescence which lagged a simultaneous increase in oxygen uptake. ATP production and oxygen uptake also varied with metabolic state. The decrease in oxidative phosphorylation following transition from aerobic to anaerobic metabolism was found to be only partially compensated by the concomitant increase in substrate-level phosphorylation, as shown by decreases of 35-52% in calculated total specific ATP production rates. The specific oxygen uptake rate decreased by 6-38% following glucose pulses of between 0.2 and 0.5 g/L, respectively, and by 50% following glutamine depletion. Subsequent pulsing of glutamine after depletion caused oxygen uptake to increase by 50%.     

Determination of intracellular potassium ion concentration in isolated rat ventricular myocytes

The sarcoplasmic potassium concentration of a suspension of rat ventricular myocytes, prepared by collagenase-induced disruption of the myocardial mass, was determined by a null-point technique. Addition of digitonin resulted in a release of potassium from the cells which was interpreted as a flux from the sarcoplasm. The intracellular potassium concentration was estimated to be 113 +/- 6mM.     

Adult ventricular myocytes isolated from CHF 146 and CHF 147 cardiomyopathic hamsters

Single ventricular myocytes have been isolated from normal and cardiomyopathic hamsters using a collagenase method. The procedure produces calcium-tolerant myocytes that are viable and appear on light and electron microscopic examination to be healthy. These cells respond to electrical stimulus and have normal intracellular resting and action potentials.     

Distribution of mitochondrial NADH fluorescence lifetimes: steady-state kinetics of matrix NADH interactions

The lifetimes of fluorescent components of matrix NADH in isolated porcine heart mitochondria were investigated using time-resolved fluorescence spectroscopy. Three distinct lifetimes of fluorescence were resolved: 0.4 (63%), 1.8 (30%), and 5.7 (7%) ns (% total NADH). The 0.4 ns lifetime and the emission wavelength of the short component were consistent with free NADH. In addition to their longer lifetimes, the remaining pools also had a blue-shifted emission spectrum consistent with immobilized NADH. On the basis of emission frequency and lifetime data, the immobilized pools contributed >80% of NADH fluorescence. The steady-state kinetics of NADH entering the immobilized pools was measured in intact mitochondria and in isolated mitochondrial membranes. The apparent binding constants (K(D)s) for NADH in intact mitochondria, 2.8 mM (1.9 ns pool) and >3 mM (5.7 ns pool), were on the order of the estimated matrix [NADH] (approximately 3.5 mM). The affinities and fluorescence lifetimes resulted in an essentially linear relationship between matrix [NADH] and NADH fluorescence intensity. Mitochondrial membranes had shorter emission lifetimes in the immobilized poo1s [1 ns (34%) and 4.1 ns (8%)] with much higher apparent K(D)s of 100 microM and 20 microM, respectively. The source of the stronger NADH binding affinity in membranes is unknown but could be related to high order structure or other cofactors that are diluted out in the membrane preparation. In both preparations, the rate of NADH oxidation was proportional to the amount of NADH in the long lifetime pools, suggesting that a significant fraction of the bound NADH might be associated with oxidative phosphorylation, potentially in complex 1.     

Correction for inner filter effects in turbid samples: fluorescence assays of mitochondrial NADH

Fluorescentdeterminations of NADH in porcine heart mitochondria were subject tosignificant errors caused by alterations in inner filter effects duringnumerous metabolic perturbations. These inner filter effects wereprimarily associated with changes in mitochondrial volume andaccompanying light scattering. The observed effects were detected in astandard commercial fluorometer with emission orthogonal to theexcitation light path and, to a lesser extent, in a light path geometrydetecting only the surface fluorescence. A method was developed todetect and correct for inner filter effects on mitochondrial NADHfluorescence measurements that were independent of the optical pathgeometry using an internal fluorescent standard and linearleast-squares spectral analysis. A simple linear correction with theinner fluorescence reference was found to adequately correct for innerfilter effects. This approach may be useful for other fluorescenceprobes in isolated mitochondria or other light-scattering media.