Development of New Calcium-Exit Blockers for Improving the Cardiac Contractility

Prof. D. Khananshvili
Department of Physiology and Pharmacology,
Tel Aviv University Medical School

This progress report is submitted by Prof. D. Khananshvili (Department of Physiology and Pharmacology, Tel Aviv University Medical School) to the Elizabeth and Nicholas Slezak Super-Center for Cardiac Research.

Subject and Goal
The cardiac sarcolemma (cell membrane) Na⁺ -Ca²⁺ exchanger mediates an electrogenic exchange (3 Na⁺ :Ca²⁺) providing a voltage-sensitive extrusion of 90-95% calcium that has entered the cardiac cell via the Ca²⁺-channels at the initial stages of action potential (1-4). A lack of selective inhibitors is still a major obstacle for understanding the molecular and cellular mechanisms in different tissues and for rational design of new drugs and molecular probes. No drug is yet available for modulating the intracellular calcium by interacting with the Na⁺ -Ca²⁺ exchanger. However, the exchanger can be indirectly modulated by some antiarrhythmic drugs (e.g. digoxin, the inhibitor of Na⁺, K⁺-ATPase), when the transmembrane Na⁺-gradient is reduced (by increasing the cytosolic concentrations of Na⁺) resulting a decline of Na⁺ -Ca²⁺ exchange and elevation of intracellular Ca²⁺. Although this intervention has a wanted positive intropic effect on heartbeat, the elevation of intracellular sodium can upset a primary ion homeostasis, thereby promoting some serious side effects on the cellular and systemic levels. We assumed that a direct pharmacological targeting of Na⁺ :Ca²⁺ exchange may increase intracellular calcium concentrations without altering intracellular sodium and potassium concentrations. This strategy may have a primary significance for practical therapy, but this pharmacological intervention is hampered, because no lead structure is yet available for inhibition of Na⁺ :Ca²⁺exchange.

The goal of this proposal was to initiate a development of a cell-permeable peptide blocked for the cardiac cell membrane (sarcolemma) sodium-calcium exchanger. It was expected that this project will generate a set of conformationally constraint, small peptides and peptidomimetic molecules that would be able to control the cardiac strength and/or rate of heartbeat by regulating the Ca²⁺-exit pathway. Our working hypothesis was that the Na⁺ :Ca²⁺ exchange blocker (NCX blocker) would elevate the transient levels of cytosolic calcium during the action potential without altering the resting levels of calcium, sodium and ion homeostasis, in general.

Background
In 1995 we designed a principally new class of cyclic hexapeptide blockers which inhibit the cardiac sarcolemma Na⁺ :Ca²⁺ exchanger activity and its partial reactions (5). Structure activity studies suggest that the mode of peptide cyclization (e.g. Cys-Arg-Cys), as well as the peptide size and positive charges, determine the inhibitory potency of the synthetic peptide (5,8). The most potent blocker, Phe-Arg-Cys-Arg-Cys-Phe-CONH2 (FRCRCFa) interacts with a cytosolic side of the membrane, resulting in a complete inhibition in regard to extravesicular (cytosolic) Ca²⁺, while the FRCRCFa induced inhibition is very rapid (<20msec)5,6). A high inhibitory potency (IC₅₀ = 20 nM) of FRCRCF has been detected in intact myocytes, suggesting that the nanomolar concentrations of the blocker may have a potent pharmacological effect (7). We found that specific parts of the FRCRCFa molecule are responsible for pharmacological effects (8). Most importantly, the tested concentrations of FRCRCFa have no detectable effects on the major ion currents of action potential, suggesting that this is a selective inhibition of the exchanger does not affect the ion-channel activities (7). These findings provide a good basis for a systematic development of better peptide inhibitors.

The main object of the future project is to provide a useful lead structre for future drug design. Our primary goal was to identify a lead structure for developing a membrane permeable inhibitor of Na⁺ :Ca²⁺ exchange. It was assumed that this kind of blocker would be accessible to the cytosolic side of the cell membrane when the blocker is added to the extracellular bath.

Current Progress
In order to introduce a cell permeable peptide blocker of the exchanger, the N-myristyl derivative of FRCRCFa was designed, synthesized and tested on the exchanger activity in myocytes. Experiments were performed using isolated rabbit right ventricular myocytes and whole-cell pat-clamp at 35-37°C (9). In this study the Na⁺ :Ca²⁺ exchange current (I_Na-Ca) L-type calcium current (I_Ca,L), inward rectifier potassium current (I_K1) and delayed rectifier potassium current (I_K) were compared in untreated cells and cells incubated in a solution containing N-myristylated FRCRCFa. We found that a 15-min pre-incubation of myocytes with 20µM N-myristyl FRCRCFa reduced the I_Na-Ca current by 30-40%, while 1h pre-incubation of the same concentration of the peptide blocker reduced the I_Na-Ca current by 60-70% as compared to the control in the absence of the peptide blocker (9). Under selective recording conditions for I_Ca,L there was little difference in I_Ca,L density between untreated and cells incubated with Myr-FRCRCFa. A Boltzmann fit to the ICa,L/V relation showed no significant alteration of half-maximal activation potential or slope factor of activation. IK1was also largely unaffected by pre-incubation of cells with Myr-FRCRCFa. I_K measured as deactivating tail current following 1-s test depolarization to a range of test potentials, was also not significantly altered by Myr-FRCRCFa. Therefore, the suppression of I_Na-Ca current in cells pre-incubated with Myr-FRCRCFa suggests that addition of Myristyl group to the N-terminal of the parent peptide conveys cell permeancy to the peptide blocker. This means that Myr-CRCRCFa applied externally to rabbit ventricular myocytes is moderately effective as an INa-Ca blocker. This cell-permeable peptide blocker seems to be reasonably selective because the major currents of action potential I_Ca,L, IK and I_K1 were largely unaffected by Myr-FRCRCFa. N-Myristylation of such conformationally constrained peptide may, therefore, provide a means of producing cell-permeant inhibitors of the cardiac Na/Ca exchanger. Despite this progress one should mention some disadvantages that limit the physiological and clinical tests of Myr-FRCRCFa.

First, Myr-FRCRCFa is expensive to synthesize and use, even in small quantities ($500/milligram). Secondly, this compound has solubility problems, which makes it difficult to perform physiological experiments. These limitations, however, should be considered in an appropriate context. Our data clearly shows that N-myristylation can confer cell permeance to short peptide sequences, which allow them to act on the intracellular face membrane-bound proteins when externally applied. Further modifications to peptides related to FRCRCFa may produce second generation peptides which may exert a more rapid and profound block from external application.

References

1. Khananshvili, D. (1998), Ion Pumps-'Adv, Molec. and Cell Biol. A23, 309-356.
2. Khananshvili, D., Price, D., Greenberg, M. and Sarne, Y. (1993) J. Biol. Chem. 268, 200-205.
3. Khananshvili, D., Shaulov, G. and Weil-Maslansky, E. (1995) Biochemistry 33, 10290-10297
4. Khananshvili, D., and Weil-Maslansky, E. (1994) Biochemistry 33: 312-319.
5. Khananshvili, D., Shaulov, G., Weil-Maslansky, E. and Baazov, D. (1995) J. Biol. Chem. 270: 16182-16188.
6. Khananshvili, D., Baazov, D., Weil-Maslansky, E., Shaulov, G. and Mester, B. (1996) Biochemistry 35: 15933-15940.
7. Hobai, I.A., Khananshvili, D. and Levi A.J. (1997) Plugers Archiv. 433: 455-463.
8. Khananshvili, D., Mester, B., Saltoun, M., Shaulov, G. and Baazov, D. (1997) Molec. Pharmacol. 51: 126-131.
9. Convery, M.K., Levi, A.J., Khananshvili, D. and Hancox, J.C. (1998) Plugers Archiv. 436: 581-590.