Research Synopsis#

Overview: The majority of my research has involved studying the control of contraction of cardiac muscle and has been undertaken at the universities of Oxford, Cambridge, London and Liverpool & Manchester. Of particular interest have been the processes which control intracellular calcium concentration. The overall approach is to take an integrated view at a cellular level of how the various transporters and channels work together to produce stable control of Ca and how this is upset in disease. I currently head a research group of around 15 people. The work has been funded from a variety of sources including Research Councils and the Wellcome Trust. Major core support presently comes from the British Heart Foundation in the form of a Personal Chair.

Studies on sodium and sodium-calcium exchange. My initial work involved a study of the Na-K pump and its effects on contraction. We demonstrated that the increase of contraction and arrhythmias produced by decreasing extracellular potassium concentration were due to inhibition of the Na-K pump. In subsequent work we obtained the first direct measurements of the electrogenic Na-K pump current in cardiac muscle and used this to further study the mechanism of the relationship between the activity of the Na-K pump and contraction. This was followed by direct measurement of intracellular sodium concentration [Na+]i using ion-selective microelectrodes. We showed that [Na+]i was decreased by depolarization an effect which (see below) was later shown to result from the effects of membrane potential on the Na-Ca exchange. The direct measurements of [Na+]i also gave the first information concerning the relationship between [Na+]i and contraction. Specifically, we found that contraction was a steep power function of [Na+]i. This was true for both phasic and tonic contractions. This, and the dependence of tension on membrane potential were interpreted in terms of cellular calcium content being controlled by a surface membrane Na-Ca exchange.
As well as the above studies on the Na-K pump in cardiac muscle, I have also studied the basic properties of the pump. This included a kinetic analysis of the interactions between ATP, Pi and potassium. In subsequent work we examined the effects of membrane potential on both the electrogenic current and the sodium fluxes produced by the pump.

Calcium oscillations and waves in the heart. My next series of experiments and publications involved studies of the control of [Ca2+]i using Ca-sensitive indicators. We investigated the factors that controlled resting [Ca2+]i. Depolarization was shown to produce a transient increase of resting [Ca2+]i which decayed over a few minutes. The transient nature of this response was analyzed in terms of the effects expected from a Na-Ca exchange. At this time we also obtained the first measurements of Ca oscillations due to spontaneous sarcoplasmic reticulum (SR) Ca release. This spontaneous Ca release is responsible for initiating some ventricular arrhythmias associated with Ca overload.

Ischaemia and cardiac metabolites. As well as studying normal aspects of cardiac function, I have also been interested in the effects of ischaemia and metabolic inhibition. Using nuclear magnetic resonance (nmr) we showed that the depressant effect of hypoxia on contraction was due to changes of intracellular phosphate rather than pH and also studied the relationship between metabolism, contraction and pH during ischaemia.

Control of intracellular Calcium. Since the mid 1980’s my work has mainly used single cells as an experimental model. This was initially used to continue our studies of metabolic inhibition. Our early work with this technique produced the first use of “caged” calcium in intact cardiac cells and demonstrated the importance of calcium-induced calcium release in cardiac muscle. In a series of subsequent papers we have used caffeine as a tool to investigate excitation-contraction coupling, in part making use of our observation that the Ca indicator Indo-1 could be used to measure caffeine as well as calcium concentration. We showed that the effects of low concentrations of caffeine could be attributed to a sensitization of calcium-induced Ca release. This and recent experiments using tetracaine to depress calcium-induced Ca release showed that manipulating calcium-induced Ca release only produces a transient effect on contraction. This shows that calcium-induced Ca release is not a useful locus for contractile regulation and suggests that, in order to produce a useful increase of contraction, the SR Ca content must be increased. These arguments have implications for many studies in which it has been suggested that calcium-induced Ca release may be modulated.

What controls the Calcium store? Recent work has been aimed at investigating the factors which control the magnitude of contraction produced by the heart and, in particular, the question of the role of the sarcoplasmic reticulum. We developed a method to obtain a quantitative measurement of the SR Ca content and showed that this could be used to measure reproducibly even the small changes of SR Ca content which accompany changes of stimulation rate. Furthermore, we were able to show that the measured changes of SR Ca content could be quantitatively accounted for by the measured Ca influx into the cell (on the L-type Ca current) and efflux (on the Na-Ca exchange).We have also applied these techniques to studying SR Ca balance during spontaneous Ca release. We have shown that, as a cell is progressively overloaded with Ca, the SR Ca content increases until a maximum level is reached at which spontaneous Ca release occurs. Further increase of Ca entry simply increases the frequency at which spontaneous Ca release occurs. We have also been interested in the factors which determine whether or not an increase of [Ca2+]i propagates. We showed, surprisingly at the time, that a locally-evoked increase of [Ca2+]i does not propagate unless the preparation is Ca-overloaded. Subsequent work showed that propagation depended on an increase of the amount of Ca released.

Stability of control of SR content. The normal control of cardiac contraction requires that the SR Ca content be regulated. We have a major interest in aspects of this regulation. We discovered that a major mechanism is a process that we have termed “autoregulation” in which changes of SR Ca content modify the amplitude of the systolic Ca transient and indirectly modify the influx or Ca into the cell (on the L-type Ca current) and efflux (on Na-Ca exchange). Much of our current research is focused on the idea that this control system can become unstable and that such instabilities may contribute to conditions such as pulsus alternans where the amplitude of the cardiac contraction (and the underlying heart beat) alternate from beat to beat.

The origin of calcium-dependent arrhythmias including CPVT. In recent work we have investigated why mutations in the Ryanodine Receptor (RyR) result in Ca waves and arrhythmias such as CPVT (catecholaminergic polymorphic ventricular tachycardia). We have found that simply modifying the properties of the RyR does not by itself produce arrhythmogenic Ca waves. Waves only occur when SR Ca is elevated thus explaining why CPVT patients only have arrhythmias during beta adrenergic stimulation when SR Ca content is elevated.

Treatment of calcium-dependent cardiac arrhythmias. As mentioned above, spontaneous release of Ca from the SR contributes to the origin of cardiac arrhythmias. A major therapeutic challenge is therefore posed by the need to remove this unwanted Ca release while preserving the normal systolic release. This is made all the more important by the fact that such cardiac arrhythmias are particularly prevalent in the context of heart failure where normal systolic Ca release is already depressed. As proof of principle we have recently demonstrated that the local anaesthetic tetracaine can abolish arrhythmogenic Ca release while increasing systolic release and we are investigating the mechanisms behind this.

Smooth muscle and other tissues. In addition to the work described above, I have also studied the physiology of smooth muscle in particular the relationship between intracellular pH and Ca ions. These findings have shown the importance of the interactions between these ions on the process of contraction in smooth muscles, such as those in the uterus and vascular beds. Other cell types I have experience with include the carotid body, dorsal root ganglia, and squid axon.

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