Enhanced Ca2+ binding of cardiac troponin reduces sarcomere length dependence of contractile activation independently of strong crossbridges

Abstract

Calcium sensitivity of the force-pCa relationship depends strongly on sarcomere length (SL) in cardiac muscle and is considered to be the cellular basis of the Frank-Starling law of the heart. SL dependence may involve changes in myofilament lattice spacing and/or myosin crossbridge orientation to increase probability of binding to actin at longer SLs. We used the L48Q cardiac troponin C (cTnC) variant, which has enhanced Ca(2+) binding affinity, to test the hypotheses that the intrinsic properties of cTnC are important in determining 1) thin filament binding site availability and responsiveness to crossbridge activation and 2) SL dependence of force in cardiac muscle. Trabeculae containing L48Q cTnC-cTn lost SL dependence of the Ca(2+) sensitivity of force. This occurred despite maintaining the typical SL-dependent changes in maximal force (F(max)). Osmotic compression of preparations at SL 2.0 μm with 3% dextran increased F(max) but not pCa(50) in L48Q cTnC-cTn exchanged trabeculae, whereas wild-type (WT)-cTnC-cTn exchanged trabeculae exhibited increases in both F(max) and pCa(50). Furthermore, crossbridge inhibition with 2,3-butanedione monoxime at SL 2.3 μm decreased F(max) and pCa(50) in WT cTnC-cTn trabeculae to levels measured at SL 2.0 μm, whereas only F(max) was decreased with L48Q cTnC-cTn. Overall, these results suggest that L48Q cTnC confers reduced crossbridge dependence of thin filament activation in cardiac muscle and that changes in the Ca(2+) sensitivity of force in response to changes in SL are at least partially dependent on properties of thin filament troponin.

Fig. 1.

Normalized sarcomere length (SL) dependence of Ca²⁺ sensitivity of force and stiffness after whole cardiac troponin (cTn) exchange containing wild-type (WT) or L48Q cardiac troponin C (cTnC). A: normalized force vs. stiffness as Ca²⁺ was varied at SL 2.0 μm (□ and ▽) and 2.3 μm (■ and ▼) for trabeculae containing WT (□ and ■) and L48Q cTnC-cTn (▽ and ▼). Normalized force-pCa (B) and absolute maximal force (F_max; C) values for WT (□ and ■) and L48Q (▽ and ▼) cTnC-cTn exchanged trabeculae at SL 2.0 μm (□ and ▽) and 2.3 μm (■ and ▼) are also shown. Error bars represent ± SE and in some cases are contained within the symbol. The data were fit with the Hill equation, and corresponding fit values are included in Table 1. *P < 0.05 as compared with SL 2.0 μm.

Fig. 2.

Normalized force-pCa relationship at SL 2.0 μm ± 3% dextran (dex) for trabeculae containing WT or L48Q cTnC-cTn. A: WT cTnC-cTn at SL 2.0 (□) and SL 2.0 + 3% dextran (■). B: L48Q cTnC-cTn at SL 2.0 (▽) and SL 2.0 + 3% dextran (▼). Error bars represent ± SE and in some cases are contained within the symbol. The data were fit with the Hill equation, and corresponding fit values are included in Table 2.

Fig. 3.

Normalized force-pCa relationship at SL 2.3 μm ± 7 mM 2,3-butanedione monoxime (BDM) for trabeculae containing WT or L48Q cTnC-cTn. A: WT cTnC-cTn at SL 2.3 (□) and SL 2.3 + 7 mM BDM (■). B: L48Q cTnC-cTn at SL 2.3 (▽) and SL 2.3 + 7 mM BDM (▼). Error bars represent ± SE and in some cases are contained within the symbol. The data were fit with the Hill equation, and corresponding fit values are included in Table 2.

Fig. 4.

Rate of force redevelopment (k_tr)-pCa and k_tr-force relationships during steady-state activation for trabeculae containing WT or L48Q cTnC-cTn at SL 2.0 and 2.3 μm. A: WT (solid) and L48Q (dashed) cTnC-cTn example force traces after a slack-restretch procedure. The corresponding length trace is shown in dotted line. B: WT cTnC-cTn k_tr-pCa relationships at SL 2.0 μm (□) and 2.3 μm (■). C: L48Q cTnC-cTn k_tr-pCa relationships at SL 2.0 μm (▽) and 2.3 μm (▼). D: WT (■) and L48Q (▼) cTnC-cTn k_tr-force relationships at SL 2.3 μm. Error bars represent ± SE and in some cases are contained within the symbol.