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.

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.

Mechanoelectric feedback (transduction) in heart: concepts and implications.

It seems that one could regard mechanoelectric feedback in normal heart as an intrinsic regulatory process that modulates normal electromechanical interactions (Fig. 6, left loop). Any physiological mechano-electro-mechano perturbation is self-adjusting and homeostatic. This preserves the status quo, or the heart adapts to form a new electro-mechanoelectric situation. The position in cardiac pathology is different (Fig. 6, right loop), particularly if the disease process produces inhomogeneities. A premature ventricular contraction can be mechanically induced by several of the accepted electrophysiological arrhythmic mechanisms. Thereafter, instantaneous feedback develops within and between regional heterogeneous mechanical conditions. These non-linear recovery processes compound interacting non-linear time courses of recovery of restitution and excitability. Changes in initial loading or mechanical conditions could initiate arrhythmia. Both mechanical and electrical inhomogeneities (also diagrammed in Fig. 5) compound the situation in the intact ventricle. This would enable a milieu of altered excitability, arrhythmogenic current flow and re-entry, to sustain the arrhythmia.

Transient depolarisation and action potential alterations following mechanical changes in isolated myocardium.

The effects of induced changes in muscle length on the action potential of frog ventricular strips and cat papillary muscle have been studied. When the frog preparation was stretched near the onset of contraction, the action potential duration shortened whereas a stretch during peak activity produced minimal change. Action potentials of cat papillary muscle do not alter with stretch at any time. By contrast, release of both preparations at a time when tension was near its peak, prolonged repolarisation or produced a transient depolarisation. The ECG changes corroborated the action potential changes. The release produced a deactivation of contraction which correlated with the transient depolarisation when the contraction and potential were expressed as ratios of the undisturbed measurements. Possible explanations for the results are discussed in terms of active and passive mechanisms that can relate to mechanical and electrical phenomena simultaneously. The mechanically induced transient depolarisations are clinically relevant, for regional ischaemia produces electrical and mechanical inhomogeneities which would cause contraction-excitation feedback interactions and thus electrophysiological abnormalities.

Effect of changes in load on monophasic action potential and segment length of pig heart in situ.

There is increasing evidence that mechano-electric feedback, defined as a change in mechanical state that precedes and alters transmembrane potential, operates in a wide variety of preparations and species including man. Load reduction is becoming a common therapeutic tool in a variety of clinical settings but the electrophysiological effects of these manoeuvres is not known. In this study the effect of changes in loading conditions on the time course of ventricular repolarisation were examined in the in situ heart in 13 pigs anaesthetised with halothane. Monophasic action potentials, electrocardiograms and segment length changes were recorded from the left ventricular epicardium using suction operated devices. Afterload was decreased by intravenous infusion of sodium nitroprusside, and increased by aortic cross clamping. Infusion of sodium nitroprusside resulted in a rise in action potential duration (measured at 70% repolarisation) in all 21 infusions (mean 3.4 ms), which was linearly related to the fall in systolic left ventricular pressure (r = 0.72, p less than 0.001) and the change in minimum systolic segment length (r = 0.46, p less than 0.05), but not to the change in maximum diastolic length (r = 0.33, NS). Aortic constriction, sufficient to elevate peak systolic left ventricular pressure back to the control level, restored the changes in action potential duration to normal. In addition, there were concomitant changes in the QT interval and T wave of the epicardial ECG. These findings show that mechano-electric feedback operates in the in situ heart and has potential importance in the clinical setting where changes in systemic blood pressure may directly alter cardiac electrophysiology.

Changes in monophasic action potential duration during the first hour of regional myocardial ischaemia in the anaesthetised pig.

The effects of ischaemia on monophasic action potential duration and conformation were studied using suction electrodes on the in situ left ventricle in anaesthetised pigs. Action potentials were recorded throughout the first hour of ischaemia and their behaviour studied on a beat to beat basis by computer analysis. The natural history of action potential duration changes in the ischaemic area was based on the presence or absence of four events. Typically, ischaemic segments showed a brief increase in action potential duration after coronary ligation (event 1), followed by a rapid fall in action potential duration (event 2), a temporary, partial, spontaneous recovery of action potential duration after about 15 min (event 3), and, finally, a decay towards inexcitability of the segment (event 4). An increase in variability of action potential duration was associated with event 2, sometimes manifest as electrical alternans. Simultaneous records from control segments of non-ischaemic myocardium were relatively unchanged. Segments of myocardium bridging the cyanotic border sometimes showed the events. Non-uniform or dispersed event 3 in ischaemia, coupled with the increased beat to beat variability after event 2, could establish the conditions for ventricular arrhythmias and fibrillation.

Mechanoelectric feedback in the atrium of the isolated guinea-pig heart.

OBJECTIVES: Atrial arrhythmias are prevalent during clinically abnormal myocardial loading, e.g. when the atrium is dilated or stretched. The initiating cause of the first premature beat that leads to this arrhythmia is unclear, as are the reasons for sustaining it. One possibility is that abnormal mechanical factors induce electrophysiological changes conductive to arrhythmia via 'mechanoelectric feedback'. The aim of this study is to investigate the concept that atrial stretch modulates the electrophysiological properties of the atrium via mechanoelectric feedback, and that mechanoelectric feedback can produce atrial arrhythmias. METHODS: Guinea-pigs were humanely killed by cervical dislocation and the hearts removed and perfused with oxygenated Krebs-Henseleit solution by the Langendorff method. The heart was paced at an atrial site near the sinus node. Monophasic action potentials and electrocardiograms were recorded form the left atrium and left ventricle with suction electrodes. Transient stretch was induced by inflating a fluid-filled intra-atrial latex balloon catheter. RESULTS: Increase in atrial volume produced several significant changes in the epicardial monophasic action potentials. It produced (i) decreases in the amplitude; (ii) a decrease in duration from 62.55 to 51.95 ms measured at 50% repolarisation (10.6 +/- 3.6 ms, P < 0.05, n = 6); (iii) an increase in duration from 122.45 to 140 ms measured at 90% repolarisation (17.55 +/- 4.5 ms, P < 0.05, n = 6) --due to the presence of early afterdepolarisations. (iv) These load-induced electrophysiological changes coincided with the occurrence of arrhythmia or premature atrial beats. CONCLUSIONS: Load changes in the atrium can produce electrophysiological changes of a kind that may be relevant to clinical atrial arrhythmia.

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.

Monophasic action potentials, electrocardiograms and mechanical performance in normal and ischaemic epicardial segments of the pig ventricle in situ.

Few studies report simultaneous electrical and mechanical recordings from the epicardium of intact beating hearts in situ during ischaemia. We use suction to apply transducers and electrodes to areas of the epicardium. This interferes little with its behaviour and allows: i) free mobility over the surface; ii) simultaneous tridirectional length changes to be recorded and summed for an overall impression of mechanical behaviour, iii) detection of changes in direction of movement; iv) simultaneous recordings of monophasic action potential and epicardial ECG. During ischaemia we can detect impaired contraction with dyskinesis and a change in direction of epicardial forces. The action potential duration shortens and we have noted impaired conduction, inexitability and recordings consistent with re-entry leading to ventricular fibrillation.

Analogue computation of indices of contraction from regional measurements in intact hearts.

Computation of indices of regional myocardial contractility such as the shortening, or the work done by or on a segment of myocardium in a single cardial cycle is tedious to perform manually, and complex interfacing and software is needed for digital computation. We describe the principles of an on-line, analogue device to perform these computations . The accuracy of the machine is demonstrated, and examples of the uses of the devices are given.

Mechanical restitution during alternans in guinea pig papillary muscles.

OBJECTIVE: The aim was to investigate alternate acceleration and retardation of mechanical restitution as a possible mechanism for mechanical alternans in isolated myocardium. METHODS: Mechanical alternans was induced in papillary muscles from the right ventricles of 11 guinea pigs (200-300 g) by rapid pacing under hypothermic conditions (T = 27 degrees C). Mechanical restitution curves were constructed by measuring the force responses to stimuli applied following variable test intervals during steady state pacing. Curves were obtained under control conditions (steady state stimulation interval 3 s), and for the beats following the large and small contractions during mechanical alternans. Monoexponentials were fitted to the restitution curves. RESULTS: The mean rate constant for restitution following the large beat in alternans was found to be slightly but significantly smaller than that following the small. Both rate constants obtained during alternans were significantly larger than the control rate constant (restitution was faster in alternans). In addition, as the alternation widened, the restitution curve of the beat following the small contraction developed a higher plateau than that following the large. CONCLUSIONS: The results confirm that the small beat in alternans is followed by faster restitution than the large. This alone is insufficient to explain the observed extent of alternans. The restitution curve for the beat following the small contraction must also rise to a higher plateau. Both the amount of calcium available for intracellular release and the rate at which it is made available vary from beat to beat.

Calcium and mechanically induced potentials in fibroblasts of rat atrium.

OBJECTIVES: Electrically non-excitable cardiac fibroblasts in the sino-atrial node region are mechano-sensitive. Rhythmic contraction of adjacent myocardium, or artificial stretch of the tissue, produce a reversible change in the membrane potential: mechanically induced potentials (MIP). Stretch of normal cardiomyocytes can be associated with intracellular calcium changes. The purpose of this study is to use pharmacological interventions to investigate the possibility that stretch-induced Ca2+ entry through ion channels in the sarcolemma and Ca2+ release from internal stores play a role in MIP generation. METHODS: Isolated spontaneously contracting or artificially stretched preparations of right atrium of rat heart were superfused with physiological solutions. An intracellular floating microelectrode recorded fibroblast MIPs and was also used for injection of current. A dye, Lucifer yellow, applied through the micropipette, identified recording sites. We assessed the role of extracellular Ca2+ using EGTA in the bathing solution. For the role of intracellular Ca2+ in the generation of MIP, several substances that influence [Ca2+]i handling were applied intracellularly by diffusion from the recording microelectrode. These include: BAPTA (to chelate intracellular Ca2+); BHQ, thapsigargin and CPA (to deplete Ca2+ from intracellular stores by inhibition of the endoplasmic reticulum (ER) ATP Ca2+ pump), and caffeine and ryanodine (to induce ER Ca2+ release). RESULTS: All the pharmacological compounds which were introduced intracellulary, and EGTA applied extracellularly, decreased the amplitude of the MIP to variable degrees. Only thapsigargin induced a bi-phasic response with an initial increase in MIP amplitude, followed by a decrease. MIP duration was reduced by most interventions, exceptions being low extracellular Ca2+, BHQ and ryanodine. Short duration extracellular application of caffeine, which was added to the perfusate as a secondary contractile stimulus, partly restored the MIPs by activation of cardiac contraction. Intracellular current injection, before any intervention, linearly altered both membrane potential (Em) and MIP amplitude (Vm). Application of compounds listed above introduced non-linearity to the Em/Vm relationship. CONCLUSION: We suggest that mechanically induced Ca2+ influx, induced through stretch-activated channels in the plasma membrane, and release of Ca2+ from the endoplasmic reticulum, play key roles in the mechanism of MIP generation. Further, our results demonstrate the existence of functional ryanodine/caffeine-sensitive Ca2+ stores in cardiac fibroblasts.

Relation between monophasic action potential duration, ST segment elevation, and regional myocardial blood flow after coronary occlusion in the pig.

There is mounting interest in the use of the monophasic action potential for electrophysiological investigation of regional myocardial ischaemia. There is, however, no systematic study of the relation between the changes in monophasic action potential and regional myocardial blood flow. In this study monophasic action potential and, for comparison, epicardial electrograms were recorded by suction electrode at 14 selected sites on the anterior surface of the normal left ventricle of the intact pig heart in situ (n = 16). The monophasic action potential duration varied across the left ventricle; it was shortest close to the left anterior descending coronary artery, increased laterally across the left ventricle, and was longer at the apex than at the base. This variation was independent of regional myocardial blood flow (measured with radiolabelled microspheres). After coronary occlusion, in nine of these pigs monophasic action potential duration significantly decreased in proportion to the reduction in regional myocardial blood flow. The ST segment of the epicardial electrogram was elevated in relation to the reduction in regional myocardial blood flow but correlated less well. The reduction in regional myocardial blood flow of approximately 50% produced maximal ST elevation. The monophasic action potential could be recorded from some sites within the border zone for up to 5 h but could not be recorded from the centre of an ischaemic zone. With ischaemic episodes of this duration there was, however, a return to baseline of the elevated ST segment for all sites within the compromised region. Monophasic action potential duration, unlike epicardial electrogram ST elevation, discriminates between central ischaemic and non-ischaemic and ischaemic sites on either side of the zone with a reduced blood flow. The results suggest that monophasic action potential duration measured by suction electrode may be a simple and reliable index of transmural-epicardial myocardial ischaemia in experimental studies and in surgery.

Electrical alternans and the onset of rate-induced pulsus alternans during acute regional ischaemia in the anaesthetised pig heart.

OBJECTIVES: Electrical alternans and mechanical alternans are both associated with cardiac ischaemia and in the case of electrical alternans there is a strong link with serious ventricular arrhythmia. We elected to investigate the relationship between electrical and mechanical alternans in control and acutely ischaemic myocardium in the intact porcine heart to determine the nature of their interaction and in particular to determine if abnormal mechanical events play a role in arhythmogenesis as has been suggested in non-ischaemic preparations. METHODS: We used rapid atrial pacing to induce regional mechanical alternans and pulsus alternans before and then at 5-min intervals after the onset of acute ischaemia induced by a 30-min ligation of a diagonal branch of the left anterior descending artery. Regional mechanical activity is measured with epicardial tripodal strain gauges and regional electrical activity is measured using suction-based monophasic action potential electrodes. To test whether alternate stretching of ischaemic segments during pulsus alternans contributed to electrical alternans we simulated pulsus alternans by clamping the proximal aorta on alternate beats. RESULTS: In control areas there was a constant discordant relationship between peak systolic pressure during alternans and action potential duration. In contrast, the ischaemic areas showed electromechanical alternans that was most frequently concordant. Clamping the proximal aorta on alternate beats produced an electrical alternans in control areas but not in the ischaemic area. CONCLUSIONS: Pulsus alternans during acute ischaemia is associated with electrical alternans that can be out of phase in control and ischaemic areas. This could increase electrical dispersion which may be pro-arrhythmic.

Sympathomimetic modulation of load-dependent changes in the action potential duration in the in situ porcine heart.

AIMS: Increased sympathetic stimulation is known to be arrhythmogenic. Likewise increased loading of the myocardium can directly generate arrhythmias. The interaction between the two on the electrophysiology of the myocardium has not been investigated before. We investigated the effect of dobutamine infusion on the shortening of the monophasic action potential duration secondary to increased loading. This was investigated during steady-state pacing and during an alteration in beat-to-beat interval in the form of a restitution curve. METHODS: Pigs were anaesthetised and their hearts exposed. Monophasic action potentials and segment lengths were recorded from the anterior surface of the left ventricle. The loading of the ventricle was increased by transiently occluding the aorta. Steady-state pacing and a restitution curve were performed. Recordings were taken before and during dobutamine infusion. RESULTS: At steady state, increased loading of the heart shortened the monophasic action potential duration by a mean (+/- s.e.m.) of 4.0 (+/- 0.5) ms (P < 0.001). During dobutamine infusion this shortening of the monophasic action potential increased. Shortening of the action potential duration increased with the dose of dobutamine up to 10 micrograms/kg/min after which a plateau was reached. By comparison to control, dobutamine depressed the electrical restitution curve at short test pulse intervals did not significantly alter the plateau. Increased loading elevated the initial section of the electrical restitution curve at short test pulse intervals and depressed the plateau in both the control recordings and those taken during dobutamine infusion. Increased loading increased the amplitude of the supernormal phase of the electrical restitution curve in control recordings and those taken during dobutamine infusion. Sympathetic stimulation by dobutamine during the steady state potentiates the effect of mechanoelectric feedback on the myocardium. The effect on the restitution curve varies with test pulse interval. At short test pulse intervals the effect of sympathetic stimulation dominates with only minor antagonistic modification by increased loading. However, at longer test pulse intervals the effect of mechanoelectric feedback is equal to that of sympathetic stimulation and is synergistic with it. CONCLUSIONS: The mechanically induced changes we describe in the normal pig heart in situ are relatively small. However, they are in the right direction to possibly contribute to arrhythmia under pathological conditions where mechanical as well as electrophysiological inhomogeneity is prominent.

Myocardial liposome uptake in the early stages of myocardial infarction.

Multiply-labelled neutral or positively-charged liposomes were given iv to open-chested dogs or pigs 0.5 h or 5 h after coronary artery occlusion. Myocardial blood flow was measured by labelled microspheres. Animals were killed 3.5 h or 6 h after coronary artery occlusion and the myocardial distribution of liposomal labels was correlated with that of the microspheres. No evidence was found that liposomes are taken up preferentially by ischaemic myocardium. The results suggest that liposomes have limited potential as a means of drug delivery in myocardial infarction.

Inosine as a selective inotropic agent on ischaemic myocardium?

Intravenous infusion of inosine (15 mg.kg-1.min-1) to the open-chested pig resulted in hypotension, coronary vasodilatation and slightly increased myocardial contractility. Following coronary occlusion, the action of inosine to increase myocardial contractility was apparently selective. Regional myocardial performance of ischaemic myocardium was increased significantly relative to nonischaemic. The selectivity of inotropic action was not mimicked by glucose-insulin-potassium. It is concluded that the selectivity is multifactoral and that the inotropic, vasodilatory and metabolic actions of the nucleoside contribute to the apparent selectivity.

Characterisation of regional myocardial dynamics during mechanical alternans in heart of anaesthetised pig.

OBJECTIVE: The aim was to investigate the behaviour of regional myocardium during mechanical alternans in a multidirectional manner. METHODS: Mechanical alternans was induced in 12 anaesthetised open chested pigs by rapid atrial pacing. In contrast to previous studies, regional mechanical activity was simultaneously assessed at up to three different sites on the left ventricle using epicardial measuring devices able to provide multidirectional information on segment motion. Pressure-length loops were plotted to assess different patterns of segmental motion. The integral of pressure and length was calculated to obtain a regional work index for each beat. RESULTS: Pressure-length loops revealed profound abnormalities in segment motion and work index during regional mechanical alternans. Myocardial segments either performed alternate amounts of positive work on each beat or alternate amounts of positive and negative work on each beat. Alternating segments contracted out of phase with each other and were occasionally stretched during systole. CONCLUSIONS: The spatio-temporal heterogeneity of regional mechanical behaviour is greatly increased during mechanical alternans.

Mechanically induced changes in action potential duration and left ventricular segment length in acute regional ischaemia in the in situ porcine heart.

OBJECTIVE: The electrophysiological events accompanying early ischaemia are important. The aim of this study was to investigate mechano-electric feedback in acute regional myocardial ischaemia in the intact heart in situ by measuring the change in action potential duration in response to increased ventricular loading imposed by transient aortic occlusion. METHODS: 11 landrace pigs were anaesthetised and their hearts exposed. A pneumatically operated blood pressure clamp was placed around the aorta. Monophasic action potentials and an index of segment motion were recorded from the epicardium in and around the ischaemic area produced by a snare placed around a coronary artery. Ventricular and systemic arterial pressures were measured. An initial aortic clamp was performed during which control recordings were taken. The coronary artery was then tied and the aorta clamped for 5-10 s every 5 min for the duration of the 30 min tie. Recordings were taken from the ischaemic area and non-ischaemic areas. RESULTS: Aortic clamp before ischaemia increased intraventricular diastolic and systolic pressure and reduced action potential duration in all the areas studied (p < 0.001). During acute regional myocardial ischaemia aortic clamping resulted in significantly more shortening of the action potential in the ischaemic area after 10 min of ischaemia than in the control area (5 ms v 10 ms, p = 0.008). Over the following 20 min the degree of shortening decreased. The greater shortening at 10 min could not be attributed to changes in the end diastolic segment length or peak ventricular pressure and could thus represent a change in the expression of mechano-electric feedback by ischaemic myocardium rather than a change in loading conditions. CONCLUSIONS: During the first 30 min following a coronary artery occlusion mechano-electric feedback in the ischaemic myocardium varies with time.