Rate dependence of mechanically induced electrophysiological changes in right ventricle of anaesthetized lambs during pulmonary artery occlusion.

AIM: Mechanically induced early afterdepolarization (EAD) is morphologically similar but different in the mechanisms with drug-induced EAD, which lead to arrhythmia. Pacing suppresses the drug-induced EAD and arrhythmia, however the effect of pacing on mechanically induced EAD and arrhythmia is not clear. This study addressed this issue in right ventricle (RV) of anaesthetized lambs. METHODS: Six lambs were anaesthetized, and their hearts exposed. Nine monophasic action potential (MAP) electrodes were placed on RV apex, outflow and inflow regions, and recorded before, during, and after a 10 s occlusion of pulmonary artery at a number of pacing rates. RESULTS: Pacing significantly reduced the baseline MAP duration at 90% repolarization (MAPD90), decreased the reduction of MAPD at early repolarization at the peak of occlusion. Nonetheless, the percentage of reduction was not significantly different among them. Pacing was able to reduce the frequencies, size of mechanically induced EADs. MAPD90 at the peak of occlusion was all shortened during pacing rather than some lengthened at intrinsic rate. Therefore, the dispersion of MAPD90 at the peak of occlusion reduced from 86 +/- 6 ms at intrinsic rate to 42 +/- 4 ms at 120 beats min-1, 38 +/- 3 ms at 150 beats min-1 and 26 +/- 3 ms at 170 beats min-1. Ultimately, pacing reduced/suppressed mechanically induced premature ventricular beats. These alterations were inversely related to heart rates. CONCLUSION: Pacing reduces/suppresses both stretch-induced EADs and arrhythmia. These modulations are remarkably similar to those on other EADs by the pacing.

Right ventricular distension alters monophasic action potential duration during pulmonary arterial occlusion in anaesthetised lambs: evidence for arrhythmogenic right ventricular mechanoelectrical feedback.

Abnormal loading and distension of the right ventricle may induce arrhythmia through the process of mechanoelectrical feedback. Nonetheless, the electrophysiological effects of right ventricular distension are ill-defined and the mechanisms which underpin mechanoelectrical feedback in the right ventricle are unknown. We examined the effects of changes in right ventricular load (complete occlusion of both caval veins or the main pulmonary artery) in 14 anaesthetised lambs, instrumented with right ventricular surface electrodes and strain gauges for recording monophasic action potential and segment length, and an integrated conductance and micromanometer-tipped catheter for measurement of right ventricular pressure and volume. Caval occlusion did not alter right ventricular segment length and monophasic action potential duration. By contrast, pulmonary arterial occlusion increased the segment length and decreased the monophasic action potential duration at 25, 50 and 70% repolarisation by 29 +/- 6, 22 +/- 4 and 17 +/- 3 ms, respectively (all P < 0.01). Of the 42 pulmonary arterial occlusions, 38 were associated with early afterdepolarisations (EADs) which increased progressively in magnitude as the occlusion was maintained until, in 32, overt arrhythmia was observed. By contrast, none of the four occlusions in which EADs were not observed resulted in arrhythmia. As a result, the proportion of occlusions which resulted in arrhythmia were greater in those associated with EADs than in those which were not (P = 0.002). Right ventricular distension alters the pattern of repolarisation, precipitates early afterdepolarisations and results in a variety of ventricular arrhythmia, including ventricular tachycardia.

Simultaneous measurement of Ca2+ and cellular dynamics: combined scanning ion conductance and optical microscopy to study contracting cardiac myocytes.

We have developed a distance modulated protocol for scanning ion conductance microscopy to provide a robust and reliable distance control mechanism for imaging contracting cells. The technique can measure rapid changes in cell height from 10 nm to several micrometers, with millisecond time resolution. This has been demonstrated on the extreme case of a contracting cardiac myocyte. By combining this method with laser confocal microscopy, it was possible to simultaneously measure the nanometric motion of the cardiac myocyte, and the local calcium concentration just under the cell membrane. Despite large cellular movement, simultaneous tracking of the changes in cell height and measurement of the intracellular Ca2+ near the cell surface is possible while retaining the cell functionality.

Functional localization of single active ion channels on the surface of a living cell.

The spatial distribution of ion channels in the cell plasma membrane has an important role in governing regional specialization, providing a precise and localized control over cell function. We report here a novel technique based on scanning ion conductance microscopy that allows, for the first time, mapping of single active ion channels in intact cell plasma membranes. We have mapped the distribution of ATP-regulated K+ channels (KATP channels) in cardiac myocytes. The channels are organized in small groups and anchored in the Z-grooves of the sarcolemma. The distinct pattern of distribution of these channels may have important functional implications.

Hybrid scanning ion conductance and scanning near-field optical microscopy for the study of living cells.

We have developed a hybrid scanning ion conductance and scanning near-field optical microscope for the study of living cells. The technique allows quantitative, high-resolution characterization of the cell surface and the simultaneous recording of topographic and optical images. A particular feature of the method is a reliable mechanism to control the distance between the probe and the sample in physiological buffer. We demonstrate this new method by recording near-field images of living cells (cardiac myocytes).

Mechano-electric feedback in right atrium after left ventricular infarction in rats.

Left ventricular myocardial infarction (MI) can lead to alterations in hemodynamic load conditions, thereby inducing right atrial hypertrophy and dilatation associated with phenotypic modulation of cardiomyocytes, electrical abnormalities, rhythm disturbances, and atrial fibrillation. However, there is limited information on the electrophysiological basis for these events. We investigated whether atrial stretch in the setting of chronic MI modulates the electrophysiological properties of cardiomyocytes via "mechano-electric feedback", providing a mechanism for atrial arrhythmia after ventricular infarction. Five weeks after left ventricular MI (n=37), action potentials (AP) were measured in right atrial tissue preparations using a current clamp scheme, and compared to sham-operated rats (SO, n=10). Contractile activity was recorded at a preload of 1 mN, and sustained stretch was applied via a micrometer. In SO, stretch of 1.75 mN shortened repolarization at 50% and prolonged it at 90%. In MI, mechanically-induced electrical alterations were observed at a significantly lower level of stretch than in SO (0.19 mN). Sustained stretch in MI prolonged AP at 90% repolarization giving rise to stretch-activated depolarizations (SAD) near 90% repolarization (SAD90). When reaching threshold for premature APs, electrical phenomena similar to atrial fibrillations were seen in some preparations. Moreover, we observed APs with prolonged duration at 25%, 50%, and 90% repolarization where stretch induced SAD near 50%. Gadolinium used at a concentration to inhibit stretch-activated channels (40microM) suppressed mechanically-induced electrical events. In conclusion, increased susceptibility after MI to mechanical stretch may predispose atrial cardiomyocytes to arrhythmia. These mechano-electrical alterations are sensitive to gadolinium suggesting involvement of stretch-activated ion channels.

Mechanoelectric feedback after left ventricular infarction in rats.

BACKGROUND: Myocardial infarction can lead to electrical abnormalities and rhythm disturbances. However, there is limited data on the electrophysiological basis for these events. Since regional contraction abnormalities feature prominently in infarction, we investigated whether stretch of myocardium from the infarction borderzone can modulate the electrophysiological properties of cardiomyocytes via mechanoelectric feedback providing a mechanism for post-infarction arrhythmia. METHODS: Five weeks after experimental myocardial infarction (MI) in rats due to ligation of the left coronary artery (n = 26) or after sham operation (SO, n = 16), action potentials (AP) were measured in left ventricular preparations from the infarction borderzone. Sustained stretch was applied via a micrometer. RESULTS: Preparations from MI generated spontaneous electrical and contractile activity. Cardiomyocytes from MI had a comparable AP amplitude, a more negative resting membrane potential, and a prolonged AP duration (APD) when compared to SO. In SO, stretch of 150 microns increased the APD90. This was associated with stretch activated depolarizations near APD90 (SAD-90). In MI, significantly lower stretch, of only 20 microns, elicited SAD-90s, or SADs near APD50 (SAD-50). Stretch-induced events were suppressed by gadolinium, at a concentration (40 microM) normally used to inhibit stretch-activated channels. CONCLUSION: After MI, SADs are generated in the infarction borderzone at lower degrees of stretch. Increased sensitivity of the membrane potential of cardiac myocytes to mechanical stimuli may contribute to the high risk of arrhythmia after infarction. These SADs may involve the opening of stretch-activated channels.

Cell volume measurement using scanning ion conductance microscopy.

We report a novel scanning ion conductance microscopy (SICM) technique for assessing the volume of living cells, which allows quantitative, high-resolution characterization of dynamic changes in cell volume while retaining the cell functionality. The technique can measure a wide range of volumes from 10(-19) to 10(-9) liter. The cell volume, as well as the volume of small cellular structures such as lamelopodia, dendrites, processes, or microvilli, can be measured with the 2.5 x 10(-20) liter resolution. The sample does not require any preliminary preparation before cell volume measurement. Both cell volume and surface characteristics can be simultaneously and continuously assessed during relatively long experiments. The SICM method can also be used for rapid estimation of the changes in cell volume. These are important when monitoring the cell responses to different physiological stimuli.

Mechanosensitivity as an integrative system in heart: an audit.

This review examines a manifold of apparently loosely linked observations and mechanisms, from membrane to man, and assembles them to support the notion that mechanoelectric transduction is an integrative regulatory system in the heart. For this, the assemblage has to satisfy, at least to some extent, criteria that apply to other integrative regulatory systems such as the endocrine and nervous systems. The integrative effectors in the endocrine system are chemical linkages, circulating hormones: in the nervous system the linkage is a network of cables, nerve conduction and neurotransmitters. Mechanical integration is would be effected through mechanical machinery, cardiac contractile and hydraulic function with attendant stress and strain transmitted via "tensegrity". This can, through the cytoskeleton, begin with membrane integrins and transmit intracellularly for example via F actins to reach the rest of the membranous integrins. Further transmission to the organ is via cell-to-cell adhesion complexes and the extracellular matrix. This tensegrity facilitates integration of force and strain changes from area to area. In consequence, and analogous to the neurendocrine system, mechanoelectric transduction should, and does (1) operate at the molecular or membrane level--this would be via mechanotransducers affecting transmembrane ionic flow; (2) operate in the cell--to influence electrophysiology; (3) have a multicellular expression--e.g. mechanical distortion of one cell can raise intracellular calcium of an adjacent cell; (4) express in the intact organ--e.g. an increase in venous return hydraulically distends the sinoatrial node, steepening its pacemaker potential, thus increasing heart rate. It should also (5) demonstrate elements of a feedback system--"mechanoelectric feedback", and (6) interact with other systems--the cytoskeleton incorporates cell signalling complexes intersecting with other signal cascades. Finally, (7) it can malfunction to produce clinical abnormality--it contributes electrophysiologically to lethal cardiac arrhythmia. This anatomical and functional behaviour of mechanoelectric transduction could sanction the prospect of viewing it as analogous to the other integrative physiological systems.

Mechanical modulation of stretch-induced premature ventricular beats: induction of mechanoelectric adaptation period.

OBJECTIVE: Mechanoelectric Feedback, a mechanical intervention inducing an electrical change, is gaining credence as a cause of cardiac arrhythmia in the clinical situation. However, the precise mechanism is unknown. To elucidate this we investigated mechanical and chemical modulation of stretch-induced premature ventricular beats. METHODS: We positioned a balloon in the left ventricle of an isolated heart (New Zealand White rabbit), perfused by the Langendorff technique. Balloon inflation regularly produces premature ventricular beats. Monophasic action potentials, ECG's and pressure recordings monitored changes, during mechanical intervention. The hearts were subjected to (i) variations in the degree of preload and duration of inflation, and (ii) cytoskeletal disrupters, colchicine and cytochalasin-B. RESULTS: Mechanical dilation of the left ventricle can not only induce premature ventricular beats, but also induce a period during which premature beats cannot be re-induced on a subsequent inflation, i.e. a mechanoelectric adaptation period. The trigger for the mechanoelectric adaptation period seems to occur immediately on balloon inflation and required up to 60 s to recover. This period started with an undershoot in the diastolic component of the monophasic action potential as well as in the peak systolic pressure, with return to control levels within the period. Deflation produced an overshoot (rather than undershoot) in the monophasic action potential duration, but this also returned to control levels within the period. Changes in preload, duration of inflation and disruption of the cytoskeleton failed to modulate the mechanically induced premature beats, or the mechanoelectric adaptation period. CONCLUSIONS: Transient ventricular stretch produces arrhythmia, followed by an antiarrhythmic adaptive period. Possible mechanisms are related to a mechanical influence on stretch-activated channels, changes in ionic concentration or diffusion, or second messenger systems, which influence membrane potential. The arrhythmic adaptation does not appear to be related to the mechanical properties of the cytoskeleton. Final elucidation of the mechanism of the mechanoelectric adaptation period demonstrated, may prove important in determining the mechanism of stretch-induced premature ventricular beats and consequently arrhythmia management.

Specialized scanning ion-conductance microscope for imaging of living cells.

A specialized scanning ion conductance microscope (SICM) for imaging living cells has been developed from a conventional patch-clamp apparatus, which uses a glass micropipette as the sensitive probe. In contrast with other types of scanning probe microscope, the SICM probe has significant advantages for imaging living cells: it is most suitable for imaging samples immersed in water solutions; and since the probe senses ion current and does not need physical contact with the sample during the scan, any preliminary preparation of cells (fixation or adherence to a substrate) is unnecessary. We have successfully imaged murine melanocytes in growth medium. The microscope images the highly convoluted surface structures without damaging or deforming them, and reveals the true, three-dimensional relief of the cells. This instrument has considerable ability to operate, potentially simultaneously, in applications as diverse as real-time microscopy, electrophysiology, micromanipulation and drug delivery.

Scanning ion conductance microscopy of living cells.

Currently there is a great interest in using scanning probe microscopy to study living cells. However, in most cases the contact the probe makes with the soft surface of the cell deforms or damages it. Here we report a scanning ion conductance microscope specially developed for imaging living cells. A key feature of the instrument is its scanning algorithm, which maintains the working distance between the probe and the sample such that they do not make direct physical contact with each other. Numerical simulation of the probe/sample interaction, which closely matches the experimental observations, provides the optimum working distance. The microscope scans highly convoluted surface structures without damaging them and reveals the true topography of cell surfaces. The images resemble those produced by scanning electron microscopy, with the significant difference that the cells remain viable and active. The instrument can monitor small-scale dynamics of cell surfaces as well as whole-cell movement.

Electrode for recording direction of activation, conduction velocity, and monophasic action potential of myocardium.

Conduction velocity and recovery of excitability are central facets of reentry arrhythmias, and yet there are no satisfactory techniques for the simultaneous measurement of both from the same area of myocardium. We have developed an electrode arrangement that allows the simultaneous recording of conduction velocity, repolarization of the myocardium together with an index of dispersion, and direction of activation of the myocardium. Three silver/silver chloride electrodes were arranged in an equilateral triangle with a reference electrode at the center. From this arrangement three monophasic action potentials were recorded. From the time of arrival of the wavefront of activation at each electrode the direction of activation and conduction velocity were calculated in real time by a computer. There was a good correlation for the in vivo signals from the circular electrode and the new electrode both for conduction velocity (r = 0.99, P < 0.001) and for direction of activation (r = 0.99, P < 0.001). This new mathematical method and electrode design allows the simultaneous measurement of conduction velocity and direction and monophasic action potential, and this can give a beat-by-beat indication of wavelength and dispersion of action potential duration.

Regional workload induced changes in electrophysiology and immediate early gene expression in intact in situ porcine heart.

Cardiac remodelling and hypertrophy induced by chronic haemodynamic overload (stretch) eventually leads to a decrease in cardiac function, an increased incidence of ventricular arrhythmia and mortality. The mechanisms by which myocytes sense haemodynamic stress and activate growth signals are largely unknown. Nuclear immediate early genes may act as third messengers, converting the stretch stimulus into long-term changes of gene expression via cytoplasmic signal transduction. However, previous studies have used cell cultures and isolated hearts, neither of which are ideal models. We have developed a new in situ porcine heart model where local strain (stretch) can be applied, for several hours if required, thus allowing the comparison of changes in electrophysiology and gene expression with unstrained myocardium in the same preparation. A pneumatically controlled stretch-device was attached to a portion of the right ventricle of an anaesthetized animal using suction. Chronic stretch was applied for 30 min or 1 h. Regional loading produced (i) a transient decrease in monophasic action potential duration (3.5+/-0.8%; P<0.05), followed by (ii) an elongation by 15 min, despite maintained stretch (3.4+/-1.5%; P<0.05 compared to the pre-stretch situation). A control segment of the right ventricle did not show these changes. Northern blot analysis showed that both c-fos and c-myc were induced in the areas sampled, but they were 12-fold and three-fold higher, respectively, in stretched compared with control tissue after 30 min. Thus, prolonged regional stretch can produce complex changes in cardiac electrophysiology and increase expression of some immediate early genes. Our model may be useful for studying the cascade of events that lead to remodelling, hypertrophy, and arrhythmia.

Contribution to heart rate variability by mechanoelectric feedback. Stretch of the sinoatrial node reduces heart rate variability.

BACKGROUND: Heart rate variability is an important prognostic indicator for sudden death. An increased risk of sudden death and arrhythmia is associated with reduced heart rate variability in heart failure. In heart failure, there is also dilatation of the atria, which raises the prospect that there could be some physiological basis to possibly link heart rate variability with atrial dilatation. We therefore investigated whether sustained atrial stretch could modulate heart rate variability directly. METHODS AND RESULTS: Pigs were anesthetized and their hearts exposed. A specially built device stretched the sinoatrial node before and after vagal section and then after administration of propranolol. Stretch of the sinoatrial node decreases heart rate variability in the following ways: The standard deviation of the beat-to-beat interval decreases (4.2 to 2.6 ms; P = .004), and the high-frequency components are reduced (control, 6.5 +/- 2.2 ms2, during stretch, 1.4 +/- 0.3 ms2, P = .003). After section of both vagi, the high-frequency components are reduced by stretch of the sinoatrial node (2.8 +/- 0.9 ms2 for control versus 1.2 +/- 0.3 ms2 during stretch; P = .05). Similarly, after both vagal section and beta-blockade, stretch of the sinoatrial node reduces the high-frequency components (10.6 +/- 3.5 ms2 for control verses 3.0 +/- 1.5 ms2 during stretch; P = .01). CONCLUSIONS: We conclude that stretch of the sinoatrial node reduces high-frequency heart rate variability. This may account in part for the reduced heart rate variability seen in clinical conditions in which the right atrium is dilated, such as congestive cardiac failure.