Calcium binding kinetics of troponin C strongly modulate cooperative activation and tension kinetics in cardiac muscle

Abstract

Tension development and relaxation in cardiac muscle are regulated at the thin filament via Ca(2+) binding to cardiac troponin C (cTnC) and strong cross-bridge binding. However, the influence of cTnC Ca(2+)-binding properties on these processes in the organized structure of cardiac sarcomeres is not well-understood and likely differs from skeletal muscle. To study this we generated single amino acid variants of cTnC with altered Ca(2+) dissociation rates (k(off)), as measured in whole troponin (cTn) complex by stopped-flow spectroscopy (I61Q cTn>WT cTn>L48Q cTn), and exchanged them into cardiac myofibrils and demembranated trabeculae. In myofibrils at saturating Ca(2+), L48Q cTnC did not affect maximum tension (T(max)), thin filament activation (k(ACT)) and tension development (k(TR)) rates, or the rates of relaxation, but increased duration of slow phase relaxation. In contrast, I61Q cTnC reduced T(max), k(ACT) and k(TR) by 40-65% with little change in relaxation. Interestingly, k(ACT) was less than k(TR) with I61Q cTnC, and this difference increased with addition of inorganic phosphate, suggesting that reduced cTnC Ca(2+)-affinity can limit thin filament activation kinetics. Trabeculae exchanged with I61Q cTn had reduced T(max), Ca(2+) sensitivity of tension (pCa(50)), and slope (n(H)) of tension-pCa, while L48Q cTn increased pCa(50) and reduced n(H). Increased cross-bridge cycling with 2-deoxy-ATP increased pCa(50) with WT or L48Q cTn, but not I61Q cTn. We discuss the implications of these results for understanding the role of cTn Ca(2+)-binding properties on the magnitude and rate of tension development and relaxation in cardiac muscle.

Figure 1

Example tension traces of activation-relaxation cycle in mouse ventricular myofibrils. A, Tension traces for myofibrils exchanged with WT cTnC (black), L48Q cTnC (red), or I61Q cTnC (blue) are shown for activation from pCa 8 to pCa 3.5 by rapid solution change (first arrow), tension redevelopment resulting from a length release-restretch (see length trace below panel A), and relaxation from pCa 3.5 to pCa 8 (second arrow) at 15 °C and <5 μmol L⁻¹ P_i (due to P_i scavenging, see Methods). Passive tension is the difference between initial tension and zero tension during the length release. B, Traces normalized to maximum tension for each condition and displayed with an expanded time scale demonstrate that activation rate (k_ACT) was decreased with I61Q cTnC (blue) but not with L48Q cTnC (red). C, Expanded time-scale traces of k_ACT and tension re-development rate (k_TR) super-imposed to show both rates are the same for L48Q cTnC (red and yellow, respectively) but differ for I61Q cTnC (blue and green, respectively). D, Normalized tension traces demonstrate that during relaxation, slow phase rate (k_REL,slow) was unchanged but duration is prolonged with L48Q cTnC (red) vs. WT cTnC (black), and that fast phase rate (k_REL,fast) did not differ.

Figure 2

Activation kinetics in the presence of 0.5 mmol L⁻¹ inorganic phosphate (P_i) for control myofibrils vs. myofibrils with I61Q cTnC. A, Example traces of activation and relaxation at pCa 4.5 for WT cTnC (control; black) and I61Q cTnC (blue) with 0.5 mmol L⁻¹ P_i. Length trace below tension trace shows transient for measurement of k_TR. B, Normalized k_ACT and k_TR traces demonstrating no difference between these rates for WT cTnC (control; black and grey, respectively), but slower k_ACT vs. k_TR for I61Q cTnC (blue vs. green, respectively). C, Summary of k_ACT and k_TR rates for 6–12 myofibrils. * P < 0.02.

Figure 3

Tension and k_TR as Ca²⁺ was varied for rat trabeculae with WT, L48Q, or I61Q cTn. A, T_max for exchange groups is shown relative to pre-exchange native T_max (1.0). Control measurements with xcTn (containing D65A cTnC which does not bind Ca²⁺ at site II) reduced T_max to <10% of WT cTn, suggesting almost complete exchange of cTn. Exchange with WT or L48Q cTn maintained ~85% T_max. With I61Q cTn T_max was greatly reduced. B, Tension-pCa curves (normalized to T_max for each condition) of summarized data for WT (black circles), L48Q (white squares), and I61Q cTn (white triangles). See Table 3 for fit parameters. C, k_TR-pCa data shows increased Ca²⁺ sensitivity of k_TR (pCa₅₀) with L48Q cTn vs. WT cTn and a dramatically reduced Ca²⁺ dependence of k_TR with I61Q cTn. D, k_TR data binned by pCa value and plotted vs. tension to show k_TR dependence on cross-bridge number (relative tension). Symbols in panels C and D are the same as panel B, and the black star (D) indicates k_TR,max in I61Q cTnC-exchanged trabeculae in a low-P_i pCa 4.0 solution, which was 5.3 ± 0.6 s⁻¹ (or ~45% reduced from k_TR,max in the traditional activating solutions with ~0.5 mmol L⁻¹ contaminating P_i). * Relative T_max and k_TR,max immediately following cTn exchange (rather than at end of pCa curve).

Figure 4

Effect of 5 mmol L⁻¹ dATP on T_max, k_TR,max, and tension-pCa in trabeculae exchanged with WT, L48Q, or I61Q cTn. A, T_max and k_TR,max increased in the presence of dATP (vs. ATP) in back-to-back maximal activations (pCa 4.0). *P<0.05, **P<0.01 vs. ATP. B, Tension-pCa relationships with dATP for WT (grey circles), L48Q (grey squares), and I61Q cTn (grey triangles). Hill fit curves for ATP are shown as dashed lines for this subset of experiments, where paired comparisons were made with dATP for L48Q (left), WT (middle), and I61Q cTn (right). See text for fit values.

Figure 5

Effect of 5 mmol L⁻¹ dATP on k_TR-tension relationship in trabeculae exchanged with WT, L48Q, or I61Q cTn. In a subset of trabeculae exchanged with WT (A), L48Q (B), or I61Q cTn (C), k_TR-tension relationships were little affected as pCa was varied in the presence of ATP (A, black symbols; B, C, white symbols) or dATP (grey symbols). * Values from back-to-back maximal activations in ATP and dATP (rather than at end of pCa curve).

Figure 6

Hill fits to cardiac data (Fig. 3B) and parallel previous studies with rabbit psoas skeletal fibers with sTnC mutants that decreased (M80Q sTnC^F27W; [14]) or increased (I60Q sTnC; [15]) k_off. The data, collected under similar solution and temperature conditions, demonstrate that cardiac muscle has a lower pCa₅₀, that altering k_off has similar effects on pCa₅₀, but that n_H is greatly reduced in cardiac but not skeletal muscle.