Effects of troponin C isoform on the action of the cardiotonic agent EMD 57033

Abstract

The effects of the cardiotonic potentiator EMD 57033 on different TnC (troponin C) isoforms were investigated. Endogenous skeletal TnC was extracted from glycerinated, permeabilized rabbit psoas fibres and replaced with either purified native rabbit psoas TnC (fast TnC) or human recombinant cTnC (cardiac TnC) (3 mg/ml in relaxing solution for 30 min). In both conditions, 10 microM EMD 57033 increased maximal calcium-activated force (Pmax) and gave a leftward shift in the pCa-tension curve. With cTnC, the increase in Pmax was much greater (228%) compared with the effect seen for fast TnC (137%), which was the same as that in unextracted control fibres. When the whole troponin was replaced rather than just TnC, the effects of EMD 57033 on fibres replaced with cTn were the same as with the cTnC subunit alone, except that the force at low Ca2+ concentrations was not increased as much. If TnC was only partially extracted, it was found that the degree of extraction did not influence the effect of EMD 57033, except when force was decreased to below 10% of the pre-extraction Pmax. Dynamic stiffness was not altered by EMD 57033 in any of the preparations. The rate of tension recovery following a release-restretch method (ktr) was decreased by EMD 57033. We conclude that EMD 57033 acts by a rate-modulating effect, and that the quantitative response of this effect is dependent on the TnC isoform present.

Figure 1. Relationship between calcium concentration (expressed as pCa) and tension in glycerinated rabbit psoas fibres both for control (○, n=7) and for 10 μM EMD 57033 (●, n=5)

(a) Shows tension normalized to maximal tension in control fibres (○), and (b) shows both curves normalized to maximal tension. In all Figures, error bars denote S.E.M. All the experiments were performed at room temperature.

Figure 2. Relationship between calcium concentration (expressed as pCa) and tension in glycerinated rabbit psoas fibres with native TnC extracted and replaced by fast skeletal wild-type TnC, control (□, n=6) and with 10 μM EMD 57033 (●, n=6)

The native control curve is shown by the open circles and dashed lines (n=7). (a) Tension normalized to maximal tension in control fibres (○), and (b) all curves normalized to maximal tension.

Figure 3. Relationship between calcium concentration (expressed as pCa) and tension in glycerinated rabbit psoas fibres with native TnC extracted and replaced by recombinant hcTnC, control (△, n=9) and with 10 μM EMD 57033 (▲, n=7)

The native control curve is shown by the open circles and dashed lines (n=7). (a) Tension normalized to maximal tension in control fibres (○), and (b) all curves normalized to maximal tension.

Figure 4. Relationship between calcium concentration (expressed as pCa) and tension in glycerinated rabbit psoas fibres with whole troponin replaced by recombinant hcTn, control (△, n=3) and with 10 μM EMD 57033 (▲, n=4)

The native control curve is shown by the open circles and dashed lines (n=7). (a) Tension normalized to maximal tension in control fibres (○), and (b) all curves normalized to maximal tension.

Figure 5. Force measurements at pCa 6.2 in native and replaced fibres

Open bars denote the absence of drug. Grey bars denote the presence of 10 μM EMD 57033. Native psoas fibres, n=7. Whole hcTn replacement, n=3. hcTnC replacement, n=9.

Figure 6. Effect of 10 μM EMD 57033 on force, varying with partial extraction of TnC

Each bin represents one fibre, extracted in EDTA solution for a varying time to functionally remove some TnC. Open bars, the post-extraction force at pCa 6.2. Filled bars, the post-extraction force at pCa 6.2 in the presence of 10 μM EMD 57033. Grey-shaded bars, the post-extraction force at pCa 5.0 in the presence of 10 μM EMD 57033.

Figure 7. Stiffness response to a rapid 1% L₀ stretch plotted against the initial force (steady-state maximal calcium-activated force at pCa 5.0, data normalized to the results at pCa 5.0 in the unreplaced state in each fibre)

Open symbols denote control fibres and filled symbols denote the presence of 30 μM EMD 57033. Circles, data from native rabbit psoas fibres; triangles, data from rabbit psoas fibres replaced with hcTnC. Each point represents one experiment.

Figure 8. Effect of EMD 57033 on time for force redevelopment following a release–restretch method (k_tr)

(a) An example trace from one fibre (220404#3). The slowing effect of 30 μM EMD 57033 can clearly be seen in the lower trace. (b) A bar chart of the mean values for k_tr taken from fibres with native or cardiac-replaced TnC, and the effect of 10 μM EMD 57033.