Melvyn Lieberman

Excitation Sequences of the Atrial Septum and the AV Node in Isolated Hearts of the Dog and Rabbit

The spread of excitation wave fronts over the atrial septum of puppies, adult dogs, and rabbits was studied in vitro by extracellular measurements with a 50μ-diameter electrode. Wave front spread through the AV node of puppies and rabbits was determined, and the functional location of the junctional region between atrial muscle and the AV node was evaluated during antegrade atrial and retrograde His bundle pacing. For dogs of all ages, the pattern of spread over the septum was greatly affected by the location of the pacemaker site simulating a high or low sinus node position. For all sinus pacing positions, wave fronts spread over the crista terminalis to form a posterior input to the AV node, while the anterior septal wave fronts formed another input. No functional evidence could be found for narrow, specialized inteniodal tracts of fixed location. Rather, wave fronts spread over broad areas creating patterns of simultaneous, multiple wave fronts which corresponded in extent to the gross anatomical landmarks of the septum. Only in this fashion were the findings consistent with the idea of three general routes of intemodal conduction in the dog and two general routes in the rabbit. The position of the functional boundary between atrium and AV node could be accounted for only by overlapping of the two tissues of this region; the boundary shifted between antegrade and retrograde conduction. Antegrade wave fronts within the AV node accelerated from the superior border of the node to the exit at the His bundle; retrograde excitation wave fronts from the His bundle decelerated through the AV node. The amplitude of the atrial wave forms as well as the status of die inputs from the posterior and anterior regions of the atrial septum were found to be important factors in AV node conduction.

Slow Conduction in Cardiac Muscle: A Biophysical Model

Mechanisms of slow conduction in cardiac muscle are categorized and the most likely identified. Propagating action potentials were obtained experimentally from a synthetically grown strand of cardiac muscle (around 50μm by 30 mm) and theoretically from a one-dimensional cable model that incorporated varying axial resistance and membrane properties along its length. Action potentials propagated at about 0.3 m/s, but in some synthetic strands there were regions (approximately 100μm in length) where the velocity decreased to 0.002 m/s. The electrophysiological behavior associated with this slow conduction was similar to that associated with slow conduction in naturally occurring cardiac muscle (notches, Wenckebach phenomena, and block). Theoretically, reasonable changes in specific membrane capacitance, membrane activity, and various changes in geometry were insufficient to account for the observed slow conduction velocities. Conduction velocities as low as 0.009 m/s, however, could be obtained by increasing the resistance ( r i ) of connections between the cells in the cable; velocities as low as 0.0005 m/s could be obtained by a further increase in r i made possible by a reduction in membrane activity by one-fourth, which in itself decreased conduction velocity by only a factor of 1/1.4. As a result of these findings, several of the mechanisms that have been postulated, previously, are shown to be incapable of accounting for delays such as those which occur in the synthetic strand as well as in the atrioventricular (VA) node.Mechanisms of slow conduction in cardiac muscle are categorized and the most likely identified. Propagating action potentials were obtained experimentally from a synthetically grown strand of cardiac muscle (around 50 mum by 30 mm) and theoretically from a one-dimensional cable model that incorporated varying axial resistance and membrane properties along its length. Action potentials propagated at about 0.3 m/s, but in some synthetic strands there were regions (approximately 100 mum in length) where the velocity decreased to 0.002 m/s. The electrophysiological behavior associated with this slow conduction was similar to that associated with slow conduction in naturally occurring cardiac muscle (notches, Wenckebach phenomena, and block). Theoretically, reasonable changes in specific membrane capacitance, membrane activity, and various changes in geometry were insufficient to account for the observed slow conduction velocities. Conduction velocities as low as 0.009 m/s, however, could be obtained by increasing the resistance (r(i)) of connections between the cells in the cable; velocities as low as 0.0005 m/s could be obtained by a further increase in r(i) made possible by a reduction in membrane activity by one-fourth, which in itself decreased conduction velocity by only a factor of 1/1.4. As a result of these findings, several of the mechanisms that have been postulated, previously, are shown to be incapable of accounting for delays such as those which occur in the synthetic strand as well as in the atrioventricular (VA) node.

Cytosolic free calcium in chick heart cells its role in cell injury

The role of cytosolic free Ca2+ (Caf) in cell injury was investigated using two methods for measuring Caf in freshly disaggregated embryonic chick heart cells. The null-point method, using arsenazo III, is based on determining the extracellular Ca2+ concentration at which no net Ca2+ movement occurs when plasma membrane permeability is increased. With this technique, the null point Caf averaged 0.23 +/- 0.07 microM (n = 6) in the basal state. Using quin2, an intracellular fluorescent dye, to measure Caf a value of 0.05 +/- 0.01 microM (n = 5) was obtained. Elevation of Caf by various agents was associated with an increase in cell injury as measured by the release of the cytosolic enzyme, LDH. However, the relationship between Caf and LDH release was not a direct one under all experimental conditions, indicating that the level of Caf is not the sole determinant of cell injury.

Mitochondrial localization and characterization of 99Tc-sestamibi in heart cells by electron probe x-ray microanalysis and 99Tc-NMR spectroscopy

As the development of targeted intracellular magnetic resonance contrast agents proceeds, techniques for the quantitative analysis of the subcellular compartmentation and characterization of metallopharmaceuticals must also advance. To this end, the subcellular distribution and chemical state of hexakis (2-methoxyisobutyl isonitrile) technetium-99 (99Tc-SESTAMIBI), the ground state of the organotechnetium radiopharmaceutical used for the noninvasive evaluation of myocardial perfusion and viability by scintigraphy, has been determined by a novel application of electron probe X-ray microanalysis (EPXMA) and 99Tc-NMR spectroscopy. In cryopreserved cultured chick heart cells equilibrated in 36 microM 99Tc-SESTAMIBI, EPXMA imaging of mitochondria yielded a respiratory uncoupler-sensitive characteristic 99Tc X-ray peak representing 32.0 +/- 2.9 nmoles Tc/mg dry weight, while EPXMA of cytoplasm or nucleus showed no peak significantly greater than the threshold detectability limit of approximately 1 nmole/mg dry weight. Technetium-99 NMR spectroscopy of heart cells equilibrated with 99Tc-SESTAMIBI showed a single peak at -45.5 ppm with no evidence of significant line broadening or chemical shift compared to aqueous chemical standards, indicating that the majority of the complex exists unbound within the mitochondrial matrix. These data quantitatively demonstrate the localization of this lipophilic cationic organometallic complex within mitochondria in situ, consistent with a sequestration mechanism dependent on membrane potentials. Furthermore, this study establishes the general feasibility of combined EPXMA and NMR spectroscopy for the direct subcellular localization and characterization of metallopharmaceuticals, techniques that are readily applicable to MR contrast agents.

Synthetic Strands of Cardiac Muscle: Growth and Physiological Implication

Cardiac muscle cells obtained fronm disaggregated embryonic chick hearts were cultured on difjerentially treated oriented substrata. Subsequent cell reaggregation, growth, and attachmwent produced linearly organized strands of cardiac muscle with dimensions suitable for electrophysiological analysis. Along the strand, areas that contained few muscle cells demonstrated reduced conduction velocity and were subject to propagation failure.

F-actin modulates swelling-activated chloride current in cultured chick cardiac myocytes.

The integrity of F-actin and its association with the activation of a Cl- current (I(Cl)) in cultured chick cardiac myocytes subjected to hyposmotic challenge were monitored by whole cell patch clamp and fluorescence confocal microscopy. Disruption of F-actin by 25 microM cytochalasin B augmented hyposmotic cell swelling by 51% (from a relative volume of 1.54 +/- 0.10 in control to 2.33 +/- 0.21), whereas stabilization of F-actin by 20 microM phalloidin attenuated swelling by 15% (relative volume of 1.31 +/- 0.05). Trace fluorochrome-labeled (fluorescein isothiocyanate or tetramethylrhodamine isothiocyanate) phalloidin revealed an intact F-actin conformation in control cells under hyposmotic conditions despite the considerable changes in cell volume. Sarcoplasmic F-actin was very disorganized and occurred only randomly beneath the sarcolemma in cells treated with cytochalasin B, whereas no changes in F-actin distribution occurred under either isosmotic or hyposmotic conditions in cells treated with phalloidin. Swelling-activated I(Cl) (68.0 +/- 6.0 pA/pF at +60 mV) was suppressed by both cytochalasin B (22.7 +/- 5.1 pA/pF) and phalloidin (22.5 +/- 3.5 pA/pF). On the basis of these results, we suggest that swelling of cardiac myocytes initiates dynamic changes in the cytoarchitecture of F-actin, which may be involved in the volume transduction processes associated with activation of I(Cl).The integrity of F-actin and its association with the activation of a Cl- current ( I Cl) in cultured chick cardiac myocytes subjected to hyposmotic challenge were monitored by whole cell patch clamp and fluorescence confocal microscopy. Disruption of F-actin by 25 μM cytochalasin B augmented hyposmotic cell swelling by 51% (from a relative volume of 1.54 ± 0.10 in control to 2.33 ± 0.21), whereas stabilization of F-actin by 20 μM phalloidin attenuated swelling by 15% (relative volume of 1.31 ± 0.05). Trace fluorochrome-labeled (fluorescein isothiocyanate or tetramethylrhodamine isothiocyanate) phalloidin revealed an intact F-actin conformation in control cells under hyposmotic conditions despite the considerable changes in cell volume. Sarcoplasmic F-actin was very disorganized and occurred only randomly beneath the sarcolemma in cells treated with cytochalasin B, whereas no changes in F-actin distribution occurred under either isosmotic or hyposmotic conditions in cells treated with phalloidin. Swelling-activated I Cl (68.0 ± 6.0 pA/pF at +60 mV) was suppressed by both cytochalasin B (22.7 ± 5.1 pA/pF) and phalloidin (22.5 ± 3.5 pA/pF). On the basis of these results, we suggest that swelling of cardiac myocytes initiates dynamic changes in the cytoarchitecture of F-actin, which may be involved in the volume transduction processes associated with activation of I Cl.

Na/H exchange in cultured chick heart cells: Secondary stimulation of electrogenic transport during recovery from intracellular acidosis

Intracellular acidosis is capable of stimulating a rapid amiloride-sensitive Na/H exchange mechanism in the cell membrane of cultured chick heart cells. The sequence of changes of intracellular sodium and potassium contents during recovery from an acid load in heart cells was determined by atomic absorption spectrophotometry and correlated with electrophysiological measurements. Induction of an intracellular acid load by removal of NH4Cl from the bathing solution caused a rapid rise in sodium content that was amiloride-sensitive. Following a peak, sodium content declined concomitant with a rise in potassium content; these changes were ouabain-sensitive and corresponded with a ouabain-sensitive membrane hyperpolarization beyond the calculated potassium equilibrium potential. These observations indicate that pHi regulation in cardiac muscle, following an intracellular acid load involves extrusion of H+ by electroneutral Na/H exchange with the consequent rise in Nai stimulating the electrogenic Na/K pump to return Nai to control level. In the presence of amiloride (10(-4) M), the hyperpolarization was slower although still present: this suggests the existence of another sodium uptake mechanism which contributes to stimulation of electrogenic transport.

Growth orientation of heart cells on nylon monofilament. Determination of the volume-to-surface area ratio and intracellular potassium concentration.

SummaryA new method is described for orienting the growth of embryonic chick heart cells as thin annuli about nylon monofilament. Analytical measurements of cell water, intracellular potassium, cell volume, and cell surface area incorporate several new techniques and provide the quantitative basis for characterizing the respective cell types in the preparation. The measurements support the hypothesis that tissue culture methodology does not alter the morphological and physiological properties of cardiac muscle cells. The preparations are ideally suited for radiotracer studies since tissue mass can be increased while retaining a relatively short diffusional distance.

Different physiological mechanisms control isovolumetric regulation and regulatory volume decrease in chick embryo cardiomyocytes.

Cultured chick embryo cardiac myocytes submitted to a 180mOsm/kg hyposmotic solution swell present a regulatory volume decrease (RVD). This RVD is mediated by a Ca2+influx followed by a 40% loss of total taurine content accompanied by the loss of lesser amounts of other osmolytes. Kidney cells respond to a gradual change in osmolality by maintaining their volume at the initial level. This is termed isovolumetric regulation (IVR), which may activate regulatory processes other than those observed with sudden changes in osmolality. When cardiac myocytes were exposed to a gradual change in osmolality, they show a partial IVR which is not dependent upon extracellular Ca2+. Potassium channel blockers, quinidine and Ba2+, and the chloride channel blocker, diphenylamine‐2‐carboxylate (DPC), compromise IVR in our model. Tritiated taurine loss and total intracellular K+contents were analyzed in cultured cardiomyocytes submitted to a gradual change in osmolality. The cultured cells lost approximately 10% of their taurine and 35% of their total K+. These findings suggest that different compensatory mechanisms are activated when cells are exposed to stepwise and gradual changes in osmolality. Inorganic osmolytes (through conductive pathways) are preferentially mobilized during the physiological and/or patho‐physiological IVR situation, perhaps reflecting energetic conservation in response to a less traumatic event for the cardiac myocytes.

An early transient current is associated with hyposmotic swelling and volume regulation in embryonic chick cardiac myocytes

Hyposmotically induced changes in membrane conductance were measured in embryonic chick cardiac myocytes using conventional and perforated patch‐clamp recording techniques; simultaneous measurements of cell volume were made from the video image of the voltage‐clamped cell. Hyposmotic challenge was associated with a rapid, transient current coincident with the onset of cell swelling; cell volume subsequently recovered towards control values (regulatory volume decrease; RVD). The transient swelling‐induced current (I(swell)) reversed at +15 mV, and was not found to be carried exclusively by any single ion in the physiological solutions. I(swell) was abolished by gadolinium (Gd3+), a blocker of stretch‐activated ion channels, and was absent when the cytoskeleton was disrupted by treatment with cytochalasin B. I(swell) was also prevented when intracellular [Ca2+] was buffered with BAPTA AM. Under those experimental conditions which prevented the generation of I(swell), cell volume regulation failed so that the cells remained swollen in hyposmotic solution. Our data reveal a functional relationship between I(swell) and RVD, whereby I(swell) is a necessary prerequisite, although not exclusively sufficient, for volume recovery following cell swelling. We propose that I(swell) is an important early signalling event which activates subsequent mechanisms to regulate cell volume.