© 2000 by European Society of Cardiology
Copyright © 2000, European Society of Cardiology
A depressing future for cyclosporine?
National Heart and Lung Institute at Imperial College School of Medicine, Dovehouse Street, London SW3 6LY, UK
* Tel.: +44-207-351-8173; fax: +44-207-376-3442 v.j.owen{at}ic.ac.uk
Received 15 March 2000; accepted 20 March 2000
See article by Janssen et al. [1] (pages 99–107) in this issue.
In this issue of the journal, Janssen and colleagues [1] examine the in vitro effects of cyclosporine A (CsA) on myocardial contractile function in multi-cellular preparations obtained from human and rabbit hearts.
The effect of CsA on cardiac muscle function is currently highly fashionable following the recent flurry of data concerning its effect on the phosphatase calcineurin in the progression of cardiac muscle hypertrophy [2]. CsA bound to its major binding protein, cyclophilin A, forms a complex that inhibits the phosphatase activity of calcineurin and, in turn, blocks dephosphorylation of the transcription factor NF-AT3 (for review, see [3]). The current paper by Janssen, in conjunction with other published data, provides us with a framework on which to hypothesise the mechanism by which CsA adversely affects cardiac contractility.
The present study by Janssen uses a number of techniques to examine the role of CsA on myocardial contractility. To summarise the key findings; CsA decreased the maximum force produced by end-stage failing and non-failing human and rabbit myocardium in response to electrical stimulation and, in addition, both contraction and relaxation rates were slowed. In an attempt to elucidate the reason for this CsA-mediated depression of twitch parameters, the authors investigate the effect of CsA on calcium handling and the contractile apparatus.
To reiterate the basis of muscle contraction following an action potential, the outer membrane of the cell is depolarized, dihydropyridine (DHP) receptors or L-type calcium channels that reside in the invaginations of the outer membrane or t-tubules, ‘sense’ this change in membrane potential, resulting in a small influx of extracellular calcium. The close proximity of the calcium release channels (CRC) in the sarcoplasmic reticulum (SR) to the DHP receptors and this rise in local calcium concentrations induces further calcium release from the SR CRC. It is this release of calcium from the SR into the cytosol that activates the contractile apparatus resulting in contraction. Relaxation occurs as the SR calcium ATPase pumps the calcium back into the SR and the process is ready to begin again.
The use of the calcium-sensitive dye, aequorin, and rapid cooling contractures (RCCs), has enabled the authors to look at the calcium transients resulting from the release of calcium from the SR into the cytosol and the amount of calcium available for release within the SR, respectively. In human preparations, CsA at 10–7 M, resulted in a reduction of force by approximately 20%, and a reduction in the amplitude of the aequorin transient by approximately 30%. Unfortunately, we are not provided with calculations converting the aequorin signal to calcium concentration. Assuming that CsA does not significantly affect fluorescence and that the signal is linear, this result clearly indicates that the amplitude of the calcium transient is also decreased by the addition of CsA. The results of RCCs show a reduction in the amount of calcium released from the SR in the presence of CsA. These data clearly fit with the depressive effect of CsA on contraction and the reduced calcium transient as measured by the aequorin signal.
Using force as the means of quantifying the SR calcium content by RCC experiments makes it essential to ascertain whether CsA exerts a direct effect on either the calcium-activated maximum force or the calcium sensitivity of the contractile apparatus. A direct effect of CsA on the contractile apparatus would potentially confound the interpretation of the RCC experiments. In order to carry out such experiments, direct access to the myofilaments is required by removing or permeabilising the outer membrane of the muscle preparation. The relationship between force and increasing concentrations of calcium (pCa=–log10 [Ca2+]) was indistinguishable in the presence or absence of CsA. This null result verifies their interpretation of the RCC data that there is less calcium in the SR, which the authors suppose is due to a ‘leaky’ SR in the presence of CsA. Additional evidence to substantiate a ‘leaky’ SR comes from the fact that energy consumption in response to CsA is increased. However, this is not because contraction in the presence of CsA is less efficient, but is due to an increase in the myocytes’ basal metabolic rate. This would be the case if there were an increase in calcium leaking out of the SR and subsequently cycling back into the SR.
Banijamali et al. [4] has also examined the effects of CsA on cardiac myocytes. These workers used a system whereby they replicated the human clinical situation more closely and exposed rats to 21 days of CsA at therapeutically relevant concentrations. They then evaluated myocardial function in vitro and arrived at similar conclusions to Janssen. The observations of spontaneous contractile activity in cardiac myocytes isolated from CsA-treated animals, plus a reduction in the level of post-rest potentiation (indicating calcium accumulation in the SR) led the authors to conclude that the calcium concentration in the SR of CsA-treated rats was reduced. The similarity of the finding of Janssen to the study by Banijamali is important, particularly given that Janssen's results are obtained after just minutes of CsA treatment in vitro. In addition, using fura-2, Olbrich et al. [5] found that, in the presence of CsA, the cytoplasmic calcium content in electrically stimulated adult rat cardiomyocytes was significantly increased. If the SR Ca content is reduced, perhaps by ‘leaky’ SR CRC, then an increased cytosolic Ca level would be expected.
What mechanisms could account for the ability of CsA to impair myocardial performance by reducing the SR calcium content? Discounting, though not writing off, transcriptional/translational effects, we are left with other possibilities such as a direct effect of CsA on key calcium-handling proteins. The most likely candidates are an increased open probability of the SR CRC and/or a decreased efficiency of the SR Ca ATPase. Another possibility is that CsA alters the calcium influx by the DHP receptors modulating the signal initiating SR calcium release.
In an elegant series of experiments, Park and colleagues [6] examined the SR CRC density and SR Ca ATPase activity in myocardial homogenates from rats treated with CsA for 21 days. In a second group of experiments, homogenates were obtained from control animals and exposed directly to CsA. In neither case was CsA found to affect the activity of the SR Ca ATPase. However, prolonged (21 days) treatment, though importantly not direct exposure to CsA, resulted in reduced SR CRC density. It is perhaps not surprising that the relative abundance of the SR CRC was not altered by direct exposure to CsA, as this would require significant changes occurring to gene expression over a very short period of time. To gain a more complete understanding of the effects of CsA on the myocytes’ calcium homeostasis, it would be of interest to examine the effects CsA has on gating of the SR CRC. It may be that although an alteration in the SR CRC density cannot be the explanation for Janssen's findings, it may be the open probability of the CRC is increased or the calcium influx by the L-type calcium channels is reduced.
In summary, this interesting paper by Janssen explores a new and exciting aspect of CsA. It may well have implications for the use of this drug in the therapeutic modulation of cardiac hypertrophy. Further studies are required to clearly elucidate the mechanistic action of CsA in altering myocardial contractile function. This paper does, however, clearly demonstrate that CsA does depress cardiac contractility and that this is probably due to an alteration in calcium handling.
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References |
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- Janssen P.M.L., Zeitz O., Keweloh B., et al. Influence of cyclosporine A on contractile function, calcium handling, and energetics in isolated human and rabbit myocardium. Cardiovascular Research (2000) 47:99–107.
[Abstract/Free Full Text] - Molkentin J.D., Lu J.-R., Antos C.L., et al. A calcineurin-dependent transcriptional pathway for cardiac hypertrophy. Cell (1998) 93:215–228.[CrossRef][Web of Science][Medline]
- Sugden P.H. Signaling in myocardial hypertrophy: life after calcineurin? [comment]. Circ Res (1999) 84:633–646.
[Free Full Text] - Banijamali H.S., ter Keurs M.H., Paul L.C., ter Keurs H.E. Excitation–contraction coupling in rat heart: influence of cyclosporin A. Cardiovasc Res (1993) 27:1845–1854.[Web of Science][Medline]
- Olbrich H.G., Donck L.V., Geerts H., et al. Cyclosporine increases the intracellular free calcium concentration in electrically paced isolated rat cardiomyocytes. J Heart Lung Transplant (1993) 12:652–658.[Web of Science][Medline]
- Park K.S., Kim T.K., Kim D.H. Cyclosporin A treatment alters characteristics of Ca2+-release channel in cardiac sarcoplasmic reticulum. Am J Physiol (1999) 276(3 Pt 2):H865–H872.[Web of Science][Medline]
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