Atomic force microscopy and dissection of gap junctions.

An atomic force microscope (AFM) was used to study the structure of isolated hepatic gap junctions in phosphate-buffered saline (PBS). The thickness of these gap junctions appears to be 14.4 nanometers, close to the dimensions reported by electron microscopy (EM). When an increasing force is applied to the microscope tip, the top membrane of the gap junction can be "dissected" away, leaving the extracellular domains of the bottom membrane exposed. When such "force dissection" is performed on samples both trypsinized and fixed with glutaraldehyde, the hexagonal array of gap junction hemichannels is revealed, with a center-to-center spacing of 9.1 nanometers.

Nucleated conformational conversion and the replication of conformational information by a prion determinant.

Prion proteins can serve as genetic elements by adopting distinct physical and functional states that are self-perpetuating and heritable. The critical region of one prion protein, Sup35, is initially unstructured in solution and then forms self-seeded amyloid fibers. We examined in vitro the mechanism by which this state is attained and replicated. Structurally fluid oligomeric complexes appear to be crucial intermediates in de novo amyloid nucleus formation. Rapid assembly ensues when these complexes conformationally convert upon association with nuclei. This model for replicating protein-based genetic information, nucleated conformational conversion, may be applicable to other protein assembly processes.

Effect of lidocaine hydrochloride on membrane conductance in mammalian cardiac Purkinje fibers.

Lidocaine depresses automaticity in cardiac Purkinje fibers by decreasing the slope of slow diastolic depolarization, but the mechanisms of this effect are poorly understood. To test the proposal that the antiautomatic effect of lidocaine might be mediated by an increase in membrane potassium conductance, transmembrane voltage (V(m)) was measured in Purkinje fibers perfused with sodium-deficient Tyrode containing choline as the major cation. V(m) was varied by altering the external potassium concentration, [K](o), from 0.5 to 150 mM before and after lidocaine, 2.14 x 10(-5) M, a concentration considered equivalent to clinical plasma antiarrhythmic levels. In Purkinje fibers, resting V(m) varies linearly with [K](o) plotted on a logarithmic scale from 4 to 150 mM, approximately as predicted by the Nernst equation. At [K](o) of 0.5-2.7 mM, resting V(m) diverges from the predicted potassium equilibrium potential (V(K)) resulting in an increased driving force for the outward K(+) current (V(m) - V(K)). In choline Tyrode at [K](o) of 2.7 mM or less, lidocaine caused a significant increase in V(m), the change being a positive linear function of (V(m) - V(K)) with a P < 0.01. This effect was more striking in Purkinje fibers with a V(m) reduced by stretch. These findings imply that lidocaine increased membrane chord conductance for the potassium ion (gK).Current-voltage relationships using intracellular current pulses were performed in choline Tyrode at [K](o) of 0.5, 2.0, and 4.0 mM and, at each [K](o), lidocaine was found to increase membrane slope conductance (GK). The increase in GK was even more apparent when the current-voltage relationships in long Purkinje fibers was corrected for cable complications or when experiments were done in short Purkinje fibers. To minimize complications due to membrane rectifier properties, GK was measured using intracellular application of small hyperpolarizing current pulses as V(m) was decreased from -90 to -60 mv by increasing the [K](o) from 3 to 15 mM before and after lidocaine. Lidocaine increased the GK over this range of V(m). These results suggest that lidocaine increases membrane potassium conductance within the range of V(m) where the pacemaker potential is seen, an action which can account for its ability to suppress automaticity, and, in part, for its ability to prevent reentrant arrhythmias.

Electrocardiogram in Hyperkalemia: electrocardiographic pattern of anteroseptal myocardial infarction mimicked by hyperkalemia-induced disturbance of impulse conduction.

In a patient with renal failure and shortness of breath, Q waves transiently appeared in the right precordial leads of the electrocardiogram (ECG) during episodes of hyperkalemia, without a substantial change in mean electrical axis. With restoration of the plasma potassium level to normal, R waves reappeared in these leads. It is concluded that the transient development of Q waves in the right precordial leads during hyperkalemia resulted from a hyperkalemia-induced conduction disturbance. Hyperkalemia, by affecting conduction in Purkinje fibers of ventricular muscle, or both, disturbed the normal sequence of septal and anterior wall depolarization and resulted in an ECG pattern that mimicked that of anteroseptal myocardial infarction. Clinically, hyperkalemia-induced conduction disturbances of this type must be included in the differential diagnosis of the ECG that suggests an anteroseptal myocardial infarction.

Electrophysiological changes in the canine atrium and ventricle during progressive hyperkalaemia: electrocardiographical correlates and the in vivo validation of in vitro predictions.

The electrophysiological response to hyperkalaemia was reinvestigated in the whole dog for several reasons including: the paucity of comparative electrophysiological and electrocardiographical studies in which atrial and ventricular tissues were simultaneously investigated; the contrast between the clarity of findings in previous in vitro studies as compared with the rather conflicting results in earlier in vivo investigations; and the difficulty in validating the extrapolation from in vitro results and theory to the in vivo situation because of deficiences in the literature. Biphasic alterations in atrial and ventricular conduction times as well as excitability in response to progressively increasing hyperkalaemia were documented. The alterations in conduction times were reflected quite accurately by relevant electrocardiographical changes. Experiments in the whole animal were designed to test predictions based on membrane theory: the results offering strong support for the validity of extrapolating from the in vitro to the in vivo situation. The results of this study should help clarify and render interpretable many of the seemingly conflicting results in the literature.

The application of atomic force microscopy for the detection of microcrystals in synovial fluid from patients with recurrent synovitis.

Synovial fluid from 33 patients with inflammatory arthritis was examined with a polarized light microscope (PLM) and an atomic force microscope (AFM). Two samples were imaged with a transmission electron microscope (TEM) to determine calcium/phosphate ratios and identify microcrystals of calcium pyrophosphate dihydrate and octacalcium phosphate. Additional correlative x-ray diffraction studies were performed on several samples including purified hydroxyapatite and sodium chloride crystals. Monosodium urate, calcium pyrophosphate dihydrate, hydroxyapatite, octacalcium phosphate, and cholesterol crystals were identified with AFM. AFM images of these microcrystals revealed detailed surface topology, including lattice parameters and structural irregularities at the crystals' surface. These features were consistent with those obtained by TEM and x-ray diffraction studies. In addition, AFM images revealed that some specimens contained microcrystals that were undetected by PLM and/or TEM. These results suggest that AFM may provide a simple yet powerful technique for the detection of microcrystals in synovial fluid taken from patients with crystal-induced arthritis.

Cardiac excitability and antiarrhythmic drugs: a different perspective.

A matrix of active and passive cellular properties determines net cardiac excitability. The hypothesis of altered excitability suggests that for cardiac arrhythmias to arise, the normal matrix must be perturbed by arrhythmogenic influences to produce a proarrhythmic matrical configuration to permit rhythm disturbances caused by abnormalities of propagation, abnormal automaticity, or altered excitability. Antiarrhythmic drugs may act with one or more components of the normal or proarrhythmic matrix to normalize or to create new antiarrhythmic or, perhaps, proarrhythmic matrices. Traditionally, antiarrhythmic drug classifications have been based on predominant drug actions. These classifications have clinical and some experimental utility but fail to consider the complicated effects that pathophysiologic influences and pharmacologic actions may have on active and passive cellular properties. Cluster analysis may allow the development of new classifications of arrhythmogenesis and antiarrhythmic drugs. The matrical concept has important clinical implications and suggest strategies for treating patients with cardiac rhythm disturbances.

Basic understanding of the electrophysiologic actions of antiarrhythmic drugs. Sources, sinks, and matrices of information.

The author creates an intellectual framework consisting of key electrophysiologic principles, basic mechanisms of arrhythmogenesis, and important drug reactions that will allow the rational use of antiarrhythmic drugs. Basic principles have been emphasized because current understanding requires it.

Electrophysiologic properties of antidysrhythmic drugs as a rational basis for therapy.

The often emergent nature of life-threatening cardiac dysrhythmias, the frequent seeming "resistance" of the abnormal heart rhythm to therapy, and the commonly encountered toxicity of antidysrhythmic agents combine to make treatment of cardiac dysrhythmias one of the strictest challenges to the practicing physician. Although electrophysiologic studies have markedly increased out understanding of dysrhythmogenesis and the actions of anti-dysrhythmic drugs, these numerous investigations have provided but little assistance to the practicing physician either as an intellectual framework or as a guide to patient care. The electrophysiologic classification of the antidysrhythmic drugs presented in this paper should be acceptable both to the electrophysiologist and the clinician since it is based on alterations in basic membrane properties and correlates well with clinical realities. It serves as a guide to initial drug selection, anticipated bioelectric complications, the use of alternative drugs, and combination antidysrhythmic therapy.

Management of arrhythmias in heart failure.

The literature for coronary artery disease as well as ischemic and dilated cardiomyopathy suggests that ventricular arrhythmias and left ventricular dysfunction are independent risk factors for sudden death, but that the presence of organic heart disease provides the substrate for potentially lethal arrhythmias. Patients with a cardiomyopathy and ventricular tachycardia are at a high risk for sudden death as a group. The general risk, then, is high for the group with CHF and arrhythmias. The prognostic indices for hypertrophic cardiomyopathy are imprecise, but the risk for sudden death for the group is high in the young and remains high even among the adult survivors. Many conditions associated with CHF and its treatment may lead to arrhythmias and are potentially reversible. Most studies suggest that EPS and exercise provocation have limited power in predicting the risk to the individual patient. Therapeutically, reversible causes of arrhythmias should be sought and corrected. In general, antiarrhythmic drug therapy has been disappointing with adequate control being achieved in only about 30 per cent of patients and uncertainties about the effectiveness of such therapy in altering long-term prognosis. This is due to various causes including the inability to find an effective drug, problems with patient compliance, the failure of physicians to properly monitor drug levels, and changes in the anatomical and physiologic substrate due to disease and therapy. Surgical ablation or resection of arrhythmogenic foci is effective in selected patients. The AICD will become first-line therapy in patients at high risk for sudden death due to ventricular arrhythmias, with antiarrhythmic drugs and other approaches being used to minimize the frequency of the arrhythmias.

Effects of quinidine sulfate on the balance among active and passive cellular properties that comprise the electrophysiologic matrix and determine excitability in sheep Purkinje fibers.

Quinidine is the most commonly used drug for the chronic treatment of ventricular arrhythmias, but it may be arrhythmogenic. Much information exists concerning quinidine's effects on active properties in cardiac tissues, but virtually nothing is known of its effects on passive properties. We studied the effects of quinidine, in a clinically relevant concentration, on the balance among active and passive cellular properties that comprise the electrophysiologic matrix that determines cardiac excitability. The multiple microelectrode method of intracellular-current application and transmembrane voltage recording was used in sheep Purkinje fibers to determine strength-duration and constant current-voltage relations as well as cable properties. A rapid, on-line computerized data analysis system tracked in time the alterations in the active and passive properties relevant to excitability. In normal fibers at [K+]o = 5.4 mM, quinidine increased cardiac excitability as manifested by a decrease in the current required to attain threshold and/or a downward shift in strength- and charge-duration relations by altering passive properties despite a depressed sodium system and a slowed conduction velocity. During washout, excitability and passive properties remained altered despite a return of descriptive action potential parameters such as the resting potential, the maximum rate of rise of phase 0, overshoot, and the action potential duration to or nearly to normal. At [K+]o = 8.0 mM, quinidine could either increase or decrease excitability; net excitability depends on the balance between altered passive properties and the depressed sodium system. The results explain, in part, the antiarrhythmic actions and arrhythmogenic potential of quinidine. The data for quinidine and other antiarrhythmic drugs are interpreted in terms of the electrophysiologic matrix, which we believe has important advantages over traditional hierarchical classifications.

A matrical approach to the basic and clinical pharmacology of antiarrhythmic drugs.

In summary, the lethal cardiac arrhythmias remain a major public health problem and their treatment is a major challenge to the clinician. We possess rapidly increasing knowledge of the electrophysiologic events which underly arrhythmogenesis and the antiarrhythmic as well as the proarrhythmic actions of drugs. Much of this electrophysiologic knowledge is irrelevant to the practicing physician. While complex, we believe that the matrical approach provides the clinician with a useful intellectual framework within which to consider the actions of arrhythmogenic influences and antiarrhythmic drugs. The matrical approach is scientifically sound, reflects clinical realities, and serves as a rational guide to the treatment of cardiac arrhythmias. The traditional classifications of antiarrhythmic drugs have served a useful purpose, but they are clearly outmoded.

Scanning (atomic) force microscopy imaging of earthworm haemoglobin calibrated with spherical colloidal gold particles.

Scanning (atomic) force microscopy (SFM) permits high-resolution imaging of a biological specimen in physiological solutions. Untreated extracellular haemoglobin molecules of the common North American earthworm. Lumbricus terrestris, were imaged in NH4Ac solution using calibrated SFM. Individual molecules and their top and side views were clearly identified and were comparable with the images of the same molecule obtained by scanning transmission electron microscopy (STEM). A central depression, the presumed mouth of the hole, was detected. We analysed 75 individual molecules for their lateral dimensions. Compression varied for different molecules, presumably because of the variation of the interaction between the SFM tip and the protein molecule. Two effective heights which correspond to the heights of the points of the haemoglobin molecules first and last touched by the tip, h1 and h2, respectively, were measured for each protein and ranged between 1.58 and 16.2 nm for h1 and 1.23 and 13.6 nm for h2. The apparent diameter was measured and ranged from 44.9 to 86.6 nm (63.2 +/- 10.5 nm, n = 75), which is about twice the diameter of the molecule reported by STEM for the top view orientation. The higher the measured effective heights, the worse was the tip convolution effect. In order to determine the tip parameters (semivertical angle, curvature of radius and the cut-off height) and to calibrate images of earthworm haemoglobin molecules, spherical gold particles were scanned as standards. The tip sectional radii at distances of h1 and h2 above the tip apex were subtracted from the apparent diameter of the protein. The calibrated lateral dimension was 29.1 +/- 3.85 nm, which is close to the reported scanning transmission electron microscopy data 30.0 +/- 0.8 nm. The results presented here demonstrate that the calibration approach of imaging gold particles is practical and relatively accurate. Calibrated SFM imaging can be applied to the study of other biomacromolecules.

Atomic (scanning) force microscopy in cardiovascular research.

The promise of atomic (scanning) force microscopy (AFM) for cardiovascular research is enormous. The AFM images by using a sharp cantilever tip to sense the repulsive and attractive forces between the tip and the sample surface. The force of interaction is kept constant while raster scanning, resulting in images of the surface contours with molecular and, on hard inorganic surfaces, even atomic resolution. Movement of the cantilever in the Z plane is detected by a laser beam reflected off the cantilever to a photodiode system, a piezotube allows an X and Y raster, and a three-dimensional image results. Its capabilities include: (1) the three-dimensional imaging of membranes and biomolecules with molecular and submolecular resolution; (2) such imaging not only of dry specimens but of specimens in a physiologic solution, thereby allowing the investigation of dynamic processes in both viable biomolecules and living cells; (3) the sensing of charge and intermolecular interaction forces; (4) the chemical or biochemical modification of the cantilever tip, which allows the identification of specific structures and the measurement of specific interactions (e.g., a ligand-receptor interaction); (5) nanometer control of the position and force of the cantilever, which, in turn, allows the physical manipulation of biomolecules, the dissection of biological structures (e.g., the separation of one gap junctional hemichannel from its neighbor, thereby revealing normally inaccessible surfaces), the delivery of ligands, drugs, or other materials to specific locations, and the precise measurement of interacting forces at specific sites; and (6) the modification of the apparatus by adding complementary methodologies (e.g., magnetic resonance imaging, fluorescence microscopy, confocal microscopy, and perhaps electrophysiology). AFM, however, is only now being applied to biological research, many technical and methodologic problems exist, and a number of them are considered in this review. Little work has been done in cardiovascular research and the purpose of this review is to introduce this new and exciting approach to investigation.