The Cardiovascular Project

This is one of the major projects involving the use of the Microelectrode Array (MEA).
Even in culture (dissociated heart cells from animals), cardiac myocytes will form viable network (beating syncytium) within a couple of days. Once the cells are connected again through gap junctions, the entire layer of cells will contract spontaneously; i.e. autorhythmic.

Cultured cardiac myocytes will form a syncytium of beating network in a few days.

A pacemaker region, which drives the entire syncytium and governs its autorhythmicity, can be found in cardiac cultures.

See how a mature syncytium of heart cells beat around an MEA recording electrode.

The propagation of cardiac field potential across the entire 8 x 8 electrode substrate can be visualised.

The same propagation over the 8 x 8 grid can also be viewed in 3D.

The conduction velocity of cardiac myocytes on an MEA can be determined.


The Point Conntact Model - The strength of the recorded signal (Vj) is largely dependent on the seal conductance (gj) between the cell and the electrode.



Our Current Approach

Through significant improvement over the past few years, the present system has much higher signal-to-noise ratio and is much more reliable than our previous system. It is thus ideal for observing the electric activity of cells over a relatively long period of time. Furthermore, despite only extracellular signals are being recorded, this system is so sensitive that even changes in extracellular action potential signal shapes can be detected. (See Below)


Cardiac Field Potential and Electrophysiology

Cultured heart cells are useful for the study of cardiac pathophysiology. Indeed, isolated rat cardiac myocytes have been used as an experimental model in the study of anoxic cell injury since the early 1980s. One major advantage is that these cells  can be easily obtained from embryonic or neonatal animals, and they provide the means of studying cellular morphology, biochemistry, and electrophysio-logical characteristics of the mammalian heart. 

The effects of cardiac hypoxia are often correlated with the functionality of the cells before and after hypoxic episodes using a patch-clamp approach. There is no doubt that the patch-clamp technique can yield important information on the cellular electrophysiology of a few cells. This approach, however, cannot provide a comprehensive picture of cell-to-cell signal propagation characteristics, and continuous long-term recording is not practical - the microelectrode arrays (MEAs) may provide the answers to these problems. The rationale behind the use of MEAs is based on the
integration of multiple cells on microchips in order to detect changes of extracellular electrophysiological signals. This system enables the recording of many cells simultaneously, which is useful when a global view of a population of cells is desired as in the case of cardiac hypoxia. 
 

The Advantages of Using the MEA in Electrophysiological Research

The MEA has a number of advantages over the traditional patch-clamp technique  including:

(1) allows measurements of changes in electrophysiological activities over a long period of time;
(2) provides high resolution spatial and temporal measurements of individual cells for signal shape comparisons;
(3) provides information about interactions between electrogenic cells at different locations of the same syncytium or whole tissues in order to better understand cell-to-cell communication; and
(4) reduces the time required for experimentation since multiple cells are recorded simultaneously. The detections of extracellular field potentials using the MEA and the intracellular action potentials using the patch-clamp technique are well-correlated. As such, the MEA has been used as an alternative tool for studying different excitable tissues; e.g. heart, brain, and stomach.


Signal Shapes Identification (Cardiac Myocytes)

Examples of a variety of extracellular action potential signal shapes/components recorded by the present our MEA system in the absence of drugs. The signal in the centre of the figure resembles an ideal signal shape (see further explanation below), where all components - (1) depolarisation, (2) sodium influx, (3)calcium influx, (4) potassium efflux - are present. Signals shown in the four corners are a variety of different signal shapes with one or more of these components being diminished or enhanced. These differences are mainly due to different types of cardiac myocyte present on the respective electrode.


The Significance of the Signal Components

The extracellular signal shapes of cardiac myocytes are composed of several signal components, and the interpretations of signal shapes have been described (Yeung et al., 2007). These signal shape components are outlined as follows:

1. The fast up-spike is related to the depolarisation of the cell membrane. The amplitude of this peak is proportional to the first derivative of the time-dependent membrane voltage. 2. The fast down-spike is related to the Na+ currents through the small cleft between the membrane and the sensor surface.
3. The slow negative signal component is mainly the result of calcium (Ca2+) influx.
4. The slow positive signal is the result of the repolarising potassium (K+) efflux.







Shape Changes Correspond to the Effects of Drug

An increasing concentration of pinacidil (a potassium channel opener), which hyperpolarises the heart, causes a concentration-dependent reductions in both amplitude and duration of field potentials.



Electrophysiological Changes and Hypoxia

The purpose of this study is to establish an in vitro experimental platform of cultured cardiacmyocytes on the MEA and to use this system to monitor the electrophysiological changes of the entire syncytium due to acute hypoxia. The obtained electrophysiological observations are compared with the presently known physiological changes, such as lactate concentration, pH, and osmolarity, of the heart under hypoxia. This study aims to show that such a cell-integrated electronic system may be useful for a variety of pharmacological studies of heart.
The figure shows that the rate of change of depolarisation from 5 to 50 min. The sodium signal magnitudes reduced and their durations lengthened progressively from 5 to 50 min of hypoxia (component 2), indicating the electrochemical gradient of sodium during these periods was gradually being reduced due to hypoxic insult. Normoxic traces across the same time points are shown as comparison. Signal components (3) and (4) cannot be seen at this time scale.

Anaesthesia and Cardioprotection - A State of Having a 'Memory'

At present, we want to investigate the Involvement of Potassium Channels in Anaesthetic-Induced Preconditioning.

It is known that pretreatment with isoflurane attenuates oxidative stress induced cell death. Over the past decade, it has been shown that ischaemic cardiac injury can be substantially alleviated by exposing the heart to pharmacological agents, such as volatile anaesthetics, before occurrence of ischaemia-reperfusion. A hallmark of this preconditioning phenomenon is its ‘memory’; i.e. cardioprotective effects persist even after removal of preconditioning stimulus.
 
It has long been suspected that anaesthetic agents prolong cardiac repolarisation by blocking ion currents; however, the clinical relevance of this blockade in subjects with reduced repolarisation reserve is unknown. Anaesthetic-induced preconditioning (APC) by isoflurane decreases sensitivity of the sarcolemmal KATP channel to inhibition by adenosine 5'-triphosphate (ATP) and decreases adenosine 5'-diphosphate (ADP) sensitivity. These effects persist even after discontinuation of the anaesthetic, suggesting a possible novel factor that may contribute to the mechanism of early memory of APC.
 
The present study aims to characterise the involvement(s) of ion channels, especially potassium channels, in anaesthesia-induced cardioprotection.
 

References

Law J.K.Y., *Yeung C. K., Frisch J., Knapp S., Ingebrandt S., Rudd J. A., Chan M. (2012). Cardioprotective effects of potassium channel openers on rat atria and isolated hearts under acute hypoxia. J Phys Pharm Adv.2:41-48.
 
Law J.K.Y., *Yeung C.K., Yiu K.L., Rudd J.A., Ingebrandt S., Chan M. (2010). A study of the relationship between pharmacological preconditioning and adenosine triphosphate-sensitive potassium (KATP) channels on cultured cardiomyocytes using the microelectrode array. J Cardiovasc Pharmacol 56, 60-68.
 
Law J.K.Y., *Yeung C.K., Hofmann B., Ingebrandt S., Rudd J.A., Offenhäusser A., Chan M. (2009). The use of microelectrode array (MEA) to study the protective effects of potassium channel openers on metabolically-compromised HL-1 cardiomyocytes. Physiol. Meas. 30, 155-167.

Yeung C.K., Sommerhage F., Wrobel G., Law J.K.Y., Offenhäusser A., Rudd J.A., Ingebrandt S., Chan M. (2009). To establish a pharmacological experimental platform for the study of cardiac hypoxia using the microelectrode array. J. Pharmacol Toxicol. Meth. 59, 146-152.

Yeung C.K., Sommerhage F., Offenhäusser A., Chan M., Ingebrandt S. (2007). Drug profiling using planar microelectrode arrays. Anal Bioanal Chem. 387, 2673-2680.


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