Cardiac Cell Ionic Regulation
Abnormal Ca transients observed in cardiac myocytes which are loaded with Fluo-3
Dr Ken MacLeod, Principal Investigator
The research projects presently underway in the laboratory are aimed at investigating the processes that control cardiac cell contraction in health and disease. Investigation of these processes are fundamental to our understanding of the workings of the heart, will allow a more logical approach to therapy and, in the longer term, provide novel treatments.
Current Projects:
- The physiology of gender differences in heart function
and The effects of sex hormones on cardiac contraction - Inhibition of ACE and the regression of hypertrophy
- Regulation of intracellular calcium
- Cellular ionic homeostasis in the hypertrophic and failing heart
- Genetic modification of proteins involved in calcium regulation
Past Projects:
Current projects:
The lifetime risk of developing coronary heart disease at age 40 years is 50% for men and 33% for women. This, coupled with the observation of a lower incidence of cardiovascular disease in premenopausal women compared with males of the same age, has fuelled speculation that oestrogen and related compounds such as phytoestrogens may have physiological actions on the cardiovascular system. We have established that oestrogen and related compounds have a negative effect on heart contraction (that can be explained by a non-genomic action) by inhibition of current flow through L-type Ca channels. Work in the laboratory concentrates upon the action of oestrogen-related compounds on the heart and the physiological significance of these actions. In particular we use phytoestrogens which are naturally occurring plant-derived compounds that exert oestrogenic effects. These phytoestrogens are regularly consumed in the human diet and they seem to possess cardioprotective qualities. The full extent of their direct cardiac action, however, remains largely unknown. The aim of this work is to investigate the acute actions of phytoestrogens and related oestrogenic compounds on calcium regulation isolated cardiac myocytes. Since Ca entry is fundamental to cardiac contraction and is facilitated by the L-type Ca channel, the experiments are crucial to our understanding the action of oestrogen-related compounds on the heart and the physiological significance of such action, to elucidate the mechanisms involved.
Research in the laboratory has thus far allowed us to find that three structurally-related phytoestrogens belonging to the isoflavones class, genistein, daidzein and equol, exert markedly different actions on cell contraction and the Ca transient. Genistein produced a stimulatory effect, despite inhibiting the Ca current, suggesting an increase in the gain of excitation-contraction coupling. The underlying mechanisms could be attributed to an increase in sarcoplasmic reticulum content, impairment of Na/Ca exchange function and heightened myofilament Ca sensitivity, although there appear to be gender-dependent differences in action. The oestrogen-related compounds (raloxifene, 3a-hydroxy-tibolone and resveratrol) have been found to share a common action of Ca channel antagonism, although the overall effects on cell contraction were variable. Some acute actions appear to be mediated via the oestrogen receptor, while others were independent of the oestrogen receptor. The findings provide evidence for complex actions of phytoestrogens and related compounds on Ca regulation in the heart.
Inhibition of ACE and the regression of hypertrophy
When parts of heart muscle die (for example after a "heart attack") the rest of the muscle mass grows in an effort to compensate for the reduced amount of active muscle. Many factors are involved in this growth process and although the cells grow, their ability to contract and to regulate important intracellular ions is poorer. This is thought to be partially due to changes in gene regulation. An enzyme important in the regulation of blood pressure, angiotensin converting enzyme (ACE), is associated with this growth process. ACE inhibitors are an established treatment for high blood pressure and heart failure and have been shown to reduce mortality from heart failure and after myocardial infarction. Small amounts of ACE inhibitors appear to be able to reverse growth-induced changes to gene regulation and we have observed that this reversal is also accompanied by parallel improvement in contractility and ion intracellular regulation. The intracellular mechanism by which inhibition of the ACE system promotes better heart cell function remains unknown. We are working to elucidate the mechanism.
Regulation of intracellular calcium
We are investigating many of the processes involved in normal cardiac contraction and relaxation.
Activation of contraction: The coupling of the electrical excitation of the heart to the production of contraction (excitation-contraction coupling) involves the interaction of a number of cellular proteins involved in calcium (Ca) homeostasis. Ca influx through the sarcolemma promotes further release of stored Ca from the sarcoplasmic reticulum (SR) via the SR Ca-release channel (the ryanodine receptor (RYR)) by a process known as Ca-induced Ca release. Both fluxes of Ca combine to initiate contraction. Current understanding of the steps linking excitation to contraction is that Ca influx increases the local Ca concentration around a cluster of release channels in sufficient amounts to activate them. The number of SR Ca-release channels activated in this way is mainly, though not exclusively, determined by the size of the Ca current. The size of the current alters the probability of clusters of release channels being activated. The probability that clusters will be activated may also be dependent upon the amount of Ca stored in the sarcoplasmic reticulum (SR Ca load).
Relaxation: On a beat-to-beat basis two main systems are involved in removing Ca from the cytoplasm and so inducing relaxation. Ca is pumped back into the SR by the phospholamban (PLB)-regulated Ca ATPase (SERCA) and extruded from the cell by the sarcolemmal Na/Ca exchange. Although cardiac cells possess other systems to decrease cytoplasmic Ca concentration (namely the sarcolemmal Ca ATPase and mitochondria) these contribute less than 5% towards relaxation of a normal twitch. SERCA and Na/Ca exchange contribute about 70 and 25% respectively towards relaxation. In steady state conditions the amount of Ca leaving the cell is the same as the amount entering so that precise Ca homeostasis is achieved.
Our current understanding of the steps linking excitation and contraction is that Ca influx increases the local Ca concentration around a cluster of release channels in sufficient amounts to activate them (Figure 1).
Figure 1
Under normal conditions this Ca activation is not self-sustaining (i.e. the Ca released through one cluster of activated SR Ca channels will not evoke release from a neighboring cluster); rather, uniform Ca influx across the sarcolemma is necessary. When a large number of SR Ca-release channels are activated in this way, a large amount of Ca is discharged into the cytoplasm and contraction occurs. The amount of Ca released from the SR is determined by the Ca current. The size of the current alters the probability of Ca spark production rather than changing the size of the sparks themselves. Thus the key elementary event in cardiac excitation-contraction coupling is the release of Ca from the SR via the activated release channel. Spark frequency also appears to be dependent upon the amount of Ca stored in the sarcoplasmic reticulum (SR Ca load). Poorly loaded SR evokes fewer sparks per unit time than a more fully loaded SR and has a greatly increased probability of spark production when activated by Ca current. Thus examination of sparks and spark frequency allows conclusions to be made about SR Ca load, elementary events in the SR Ca release pathway and the coupling between excitation and contraction in the intact cardiac myocyte.
Relaxation takes place as a result of two main systems decreasing the cytoplasmic Ca concentration. The SR Ca-ATPase pumps Ca back into the SR ready for release at the next beat and the sarcolemmal Na/Ca exchange extrudes the Ca that entered the cell via the Ca channels (see Figure 2).
Figure 2
Thus cytoplasmic Ca levels in the cardiac myocyte are precisely regulated. The SR provides the link between contraction and relaxation because failure of uptake not only will slow relaxation, but also will decrease the amount of release. This is because a decrease in Ca uptake will, in turn, reduce the amount of Ca available for release by the SR at the next beat, leading to a reduced transient and so inadequate release of Ca may well be a consequence of a failing uptake or relaxation mechanism.
| Confocal images of cardiac myocytes immuno-labelled with antibody to the Na/Ca exchange. Both images are of the same cell at different magnifications and the green fluorescence highlights the presence of the exchangers in the cell membrane and the t-tubules | |
Cellular ionic homeostasis in the hypertrophic and failing heart
Hypertrophied and failing cardiac tissue contracts poorly. The findings in whole tissue can also be observed in single cells, suggesting that the poorer function exhibited by hearts nearing failure is a consequence of systems failing at the cellular level. Myocytes isolated from the hearts of patients with dilated or ischaemic cardiomyopathies, when compared with those isolated from healthy donor hearts, have Ca transients which are slower to recover, diastolic Ca levels which are higher, and peak Ca levels which are smaller. There is growing evidence that the cellular locus for poorer function is the sarcoplasmic reticulum (SR). Studies of hypertrophic and failing hearts show generally that both mRNA and protein levels of the SR Ca ATPase (SERCA 2) are reduced. There is also evidence in heart failure of reduced mRNA coding for the SR Ca-release channel. Decreased expression of such proteins could account for the poorer contraction and relaxation. Thus, in hypertrophy or in heart failure, expression of genes involved in the regulation of Ca may not follow the overall increase in gene expression that results in cardiac cell growth. This may lead to a decrease in the density of various proteins controlling Ca and contribute to the slowed removal of Ca from the cytoplasm and so contraction is slower and poorer. We are investigating the effects of hypertrophy and heart failure on the main proteins involved in Ca regulation.
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These images show the difference between |
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a normal, healthy |
an abnormal hypertrophied |
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Genetic modification of proteins involved in calcium regulation
There is evidence in heart failure of altered mRNA coding for some of the proteins involved in Ca regulation and for altered expression of these proteins. This may lead to changes in the way Ca is controlled by the cells. The aim of these studies is to alter the expression of certain Ca handling proteins and observe the changes to Ca homeostasis. For example, in collaboration with Dr. Harding's group, we have infected adult cardiac myocytes with an adenoviral vector carrying the cDNA for the Na/Ca exchange and have investigated the functional consequences.
PAST PROJECTS:
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One of the most serious complications of heart disease of any cause is the development of disturbances of cardiac rhythm. Patients suffering from most forms of heart disease have a significantly increased risk of sudden death because of a tendency to these arrhythmias. A proportion of cardiac arrhythmias may arise from triggered activity secondary to small oscillations of electrical activity (depolarizations) within the cell. These afterdepolarizations are believed to result from abnormalities of intracellular Ca regulation. We are investigating the subcellular events that lead to the generation of afterdepolarizations. These images use different methods to show cardiac arrhythmia within the same cell. It is clear that the arrhythmic cell (on the left of both images) has uncoordinated calcium movements which translate to the abnormal contraction and ultimately an abnormal heart beat. |
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ADDITIONAL COLLABORATORS:Matthew Edenbrow (Research Technician) |
