Contractile effects of the exchange of cardiac troponin for fast skeletal troponin in rabbit psoas single myofibrils

Abstract

The effects of the removal of fast skeletal troponin C (fsTnC) and its replacement by cardiac troponin C (cTnC) and the exchange of fast skeletal troponin (fsTn) for cardiac troponin (cTn) were measured in rabbit fast skeletal myofibrils. Electrophoretic analysis of myofibril suspensions indicated that replacement of fsTnC or exchange of fsTn with cTnC or cTn was about 90% complete in the protocols used. Mechanical measurements in single myofibrils, which were maximally activated by fast solution switching, showed that replacement of fsTnC with cTnC reduced the isometric tension, the rate of tension rise following a step increase in Ca2+ (kACT), and the rate of tension redevelopment following a quick release and restretch (kTR), but had no effect on the kinetics of the fall in tension when the concentration of inorganic phosphate (Pi) was abruptly increased (kPi(+)). These data suggest that the chimeric protein produced by cTnC replacement in fsTn alters those steps controlling the weak-to-strong crossbridge attachment transition. Inefficient signalling within the chimeric troponin may cause these changes. However, replacement of fsTn by cTn had no effect on maximal isometric tension, kACT or kTR, suggesting that these mechanics are largely determined by the isoform of the myosin molecule. Replacement of fsTn by cTn, on the other hand, shifted the pCa50 of the pCa-tension relationship from 5.70 to 6.44 and reduced the Hill coefficient from 3.3 to 1.4, suggesting that regulatory protein isoforms primarily alter Ca2+ sensitivity and the cooperativity of the force-generating mechanism.

Figure 2. Tension generation in rabbit psoas myofibrils following TnC extraction and replacement with exogenous fsTnC or cTnC at 15°C

Representative preparations treated with the single myofibril TnC extraction–replacement protocol. A and C, slow time base recordings of myofibril tension (upper traces) and length (lower traces) during full activation–relaxation cycles induced by fast solution switching between pCa 8.0 and 4.5. In both myofibrils, tension was recorded before (control) and after exhaustive extraction of endogenous TnC (TnC-extr). Finally, tension was recorded after reconstitution with either fsTnC (fsTnC, A) or cTnC (cTnC, C). In each contraction cycle a large release–restretch was applied to the preparation under steady-state conditions of activation to measure k_tr. k_tr values were: 8.2 and 7.9 s⁻¹ in control and fsTnC, respectively (A), and 7.6 and 3.2 s⁻¹ in control and cTnC, respectively (C). B and D, same traces as in A and C, respectively, on an expanded time base and normalized to the amplitude of the force change to illustrate the effects of TnC replacement on the kinetics of tension activation; B, k_act 8 s⁻¹ in both control and fsTnC; D, k_act 7.6 and 3.3 s⁻¹ in control and cTnC, respectively.

Figure 1. TnC and Tn replacement in rabbit psoas myofibril suspensions

In each gel the myofibrils loaded into the lane were washed of the solutions in which they had been centrifuged to form a pellet, the supernatant was removed and the pellet washed and then resuspended in rigor-EGTA solution. A, 10–20 % gradient SDS gel of untreated control myofibrils (ctrl), myofibrils that had been extracted to remove the endogenous TnC (TnC extr) and myofibrils from which endogenous TnC had been extracted and then replaced by either exogenous fsTnC (fsTnC repl) or cTnC (cTnC repl). B, 12 % SDS gel of untreated control myofibrils (ctrl), myofibrils in which the endogenous whole Tn complex had been exchanged with cardiac Tn (cTn-exch), and lanes containing only cardiac Tn (cTn) or fsTn (fsTn). C, Western blot of a gel containing cTn-exchanged myofibrils (cTn exch) shows a large amount of cardiac TnT (cTnT) in the myofibrils but less than 5 % of the native fsTnT. Thus, an almost complete replacement of endogenous fsTn has taken place. The left-most lane contains cTnT and the right-most, fsTnT.

Figure 3. Effects of partial TnC extraction without TnC replacement on tension and apparent rates of tension generation in rabbit psoas myofibrils at 15°C

A, full activation–relaxation cycles recorded from a myofibril under control conditions and after 1 min perfusion with the TnC extraction solution. In both cases, pCa was switched between 8.0 and 4.5. The drop in maximal tension induced by TnC extraction (P₀ after TnC extraction was 42 % of the control value) was not accompanied by any significant change in k_act (7.2 vs. 7.1 s⁻¹ in control vs. TnC extraction) and k_tr (7.4 vs. 7.3 s⁻¹), as better shown in B (for k_act) and C (for k_tr) on a faster time base and after normalization of the traces for the maximal tension. D, average behaviour of maximal tension (filled circles) and k_act (open circles) following progressively longer exposure of myofibrils to the extraction solution. Data points are means ±s.e.m. of six to nine myofibrils.

Figure 4. Tension transients of fsTnC- and cTnC-replaced myofibrils in response to sudden changes in [P_i] at 5°C

Representative examples from batches of myofibrils from which endogenous TnC had been extracted and then replaced by either fsTnC (A and B) or cTnC (C and D). A and C, slow time base recordings of myofibril tension (upper traces) and length (lower traces). Both myofibril types were initially fully relaxed in a P_i-free solution (pCa 8.0, [P_i] 5 μm) and then maximally activated by switching to a pCa 4.5 P_i-free solution. After a quick release–restretch was applied to the myofibrils to measure k_tr, a sudden increase in [P_i] was produced by switching to a 2 mm P_i maximally activating solution. A new release–restretch was applied to the myofibrils, after which [P_i] was suddenly reduced back to 5 μm. After a final release–restretch, the myofibrils were fully relaxed (pCa 8.0). B and D, fast time base recordings of the myofibril tension responses to sudden changes in [P_i]. k_Pi(+) and k_Pi(−) are the rate constants of the tension changes in response to sudden [P_i] increase and decrease, respectively.

Figure 5. Tension generation in rabbit psoas myofibrils at 15°C following whole Tn complex replacement by cTn

Representative recordings of tension (upper traces) and length (lower traces) from batches of control, unexchanged myofibrils (A) and myofibrils whose native fsTn had been exchanged with cTn (B). Both myofibril types were initially activated by switching the pCa from 8.0 to 6.25 and then maximally activated by a further stepwise pCa decrease to 3.5. Large release–restretches were applied to the preparations under steady-state conditions of activation to measure k_tr. Tension, k_act and k_tr measured at maximal activation appear to be the same for the cTn- and fsTn-containing myofibrils, while the larger submaximal tension developed by the cTn-containing myofibril at pCa 6.25 indicates that the Ca²⁺ sensitivity of tension is greater in the cTn-exchanged myofibril.

Figure 6. Effect of cTn replacement for fsTn on the Ca²⁺ sensitivity of tension in rabbit psoas myofibrils

pCa–tension relationships of control, fsTn-containing, myofibrils (filled circles) and cTn-replaced myofibrils (open circles). Data points are means ±s.e.m. of five to 13 myofibrils at 15°C. The continuous lines are drawn according to the parameters developed by fitting the data to the Hill equation: P/P₀= 1/(1 + 10^{(-nH(pCa50 − pCa))}); pCa₅₀= 5.70 ± 0.02 and 6.44 ± 0.04; n_H= 3.30 ± 0.49 and 1.42 ± 0.15 for the control and cTn-replaced myofibrils, respectively. Filled triangles are average results from four control experiments in which myofibril endogenous fsTn was replaced by exchange with an exogenous fsTn complex.

Figure 7. Dependence of the kinetics of tension development on the level of Ca²⁺ activation in fsTn- and cTn-containing myofibrils

Scattered k_tr values of control, fsTn-containing, myofibrils (filled circles) and cTn-replaced myofibrils (open circles) are plotted versus steady-state isometric tension at different values of [Ca²⁺] (15°C). Data points at maximal activation are means ±s.e.m. of 27–29 myofibrils (see Table 1). The dotted line is a single exponential function fitted to the data: k_tr= 1.37 + 0.0345 exp (P/P₀/0.193). Open triangles are mean k_tr values measured at maximal Ca²⁺ activation in seven cTnC-replaced myofibrils (relative tension 0.59) and in 11 control, TnC-unextracted myofibrils (relative tension = 1; see Table 1).