Effect of Ca2+ binding properties of troponin C on rate of skeletal muscle force redevelopment

To investigate effects of altering troponin (Tn)C Ca2+ binding properties on rate of skeletal muscle contraction, we generated three mutant TnCs with increased or decreased Ca2+ sensitivities. Ca2+ binding properties of the regulatory domain of TnC within the Tn complex were characterized by following the fluorescence of an IAANS probe attached onto the endogenous Cys99 residue of TnC. Compared with IAANS-labeled wild-type Tn complex, V43QTnC, T70DTnC, and I60QTnC exhibited ∼1.9-fold higher, ∼5.0-fold lower, and ∼52-fold lower Ca2+ sensitivity, respectively, and ∼3.6-fold slower, ∼5.7-fold faster, and ∼21-fold faster Ca2+ dissociation rate (koff), respectively. On the basis of Kd and koff, these results suggest that the Ca2+ association rate to the Tn complex decreased ∼2-fold for I60QTnC and V43QTnC. Constructs were reconstituted into single-skinned rabbit psoas fibers to assess Ca2+ dependence of force development and rate of force redevelopment (ktr) at 15°C, resulting in sensitization of both force and ktr to Ca2+ for V43QTnC, whereas T70DTnC and I60QTnC desensitized force and ktr to Ca2+, I60QTnC causing a greater desensitization. In addition, T70DTnC and I60QTnC depressed both maximal force (Fmax) and maximal ktr. Although V43QTnC and I60QTnC had drastically different effects on Ca2+ binding properties of TnC, they both exhibited decreases in cooperativity of force production and elevated ktr at force levels <30%Fmax vs. wild-type TnC. However, at matched force levels >30%Fmax ktr was similar for all TnC constructs. These results suggest that the TnC mutants primarily affected ktr through modulating the level of thin filament activation and not by altering intrinsic cross-bridge cycling properties. To corroborate this, NEM-S1, a non-force-generating cross-bridge analog that activates the thin filament, fully recovered maximal ktr for I60QTnC at low Ca2+ concentration. Thus TnC mutants with altered Ca2+ binding properties can control the rate of contraction by modulating thin filament activation without directly affecting intrinsic cross-bridge cycling rates

Effect of Temperature on the Structure of Trout Troponin C

Adaptation for life at different temperatures can cause changes in many aspects of an organism. One example is the expression of different protein isoforms in species adapted to different temperatures. The calcium regulatory protein cardiac troponin C (cTnC), from rainbow trout (Oncorhynchus mykiss), is a good model for studying temperature effects, both because of its low physiological temperature and because mammalian cTnC, extensively studied at higher temperatures, can be used for comparison. We determined the structure and studied the backbone dynamics of the regulatory domain of trout cardiac troponin C (ScNTnC) with one Ca2+ bound at 7 and 30 °C, using nuclear magnetic resonance spectroscopy (NMR). The overall fold of the regulatory domain of trout cTnC at both temperatures is similar to the regulatory domain of mammalian (human, bovine, and porcine isoform) cTnC bound to one Ca2+. By comparing the trout structures at the two temperatures, we identify differences between the positions of the helices flanking the calcium binding loops, and the overall structure at 7 °C is more compact than that at 30 °C. The structure at 7 °C is more similar to the mammalian cTnC, which was determined at 30 °C, indicating that they have the same conformation at their respective physiological temperatures. The dynamic properties of the regulatory domain of trout cTnC are similar at the two temperatures that were used in these studies