Structural and functional consequences of cardiac troponin C L57Q and I61Q Ca(2+)-desensitizing variants.

Two cTnC variants, L57Q and I61Q, both of which are located on helix C within the N domain of cTnC, were originally reported in the skeletal muscle system [Tikunova, Davis, J. Biol. Chem. 279 (2004) 35341-35352], as the analogous L58Q and I62Q sTnC, and demonstrated a decreased Ca(2+) binding affinity. Here, we provide detailed characterization of structure-function relationships for these two cTnC variants, to determine if they behave differently in the cardiac system and as a framework for determining similarities and differences with other cTnC mutations that have been associated with DCM. We have used an integrative approach to study the structure and function of these cTnC variants both in solution and in silico, to understand how the L57Q and I61Q mutations influence Ca(2+) binding at site II, the subsequent effects on the interaction with cTnI, and the structural changes which are associated with these changes. Steady-state and stopped flow fluorescence spectroscopy confirmed that a decrease in Ca(2+) affinity for recombinant cTnC and cTn complexes containing the L57Q or I61Q variants. The L57Q variant was intermediate between WT and I61Q cTnC and also did not significantly alter cTnC-cTnI interaction in the absence of Ca(2+), but did decrease the interaction in the presence of Ca(2+). In contrast, I61Q decreased the cTnC-cTnI interaction in both the absence and presence of Ca(2+). This difference in the absence of Ca(2+) suggests a greater structural change in cNTnC may occur with the I61Q mutation than the L57Q mutation. MD simulations revealed that the decreased Ca(2+) binding induced by I61Q may result from destabilization of the Ca(2+) binding site through interruption of intra-molecular interactions when residue 61 forms new hydrogen bonds with G70 on the Ca(2+) binding loop. The experimentally observed interruption of the cTnC-cTnI interaction caused by L57Q or I61Q is due to the disruption of key hydrophobic interactions between helices B and C in cNTnC. This study provides a molecular basis of how single mutations in the C helix of cTnC can reduce Ca(2+) binding affinity and cTnC-cTnI interaction, which may provide useful insights for a better understanding of cardiomyopathies and future gene-based therapies.

Transgenic overexpression of ribonucleotide reductase improves cardiac performance.

We previously demonstrated that cardiac myosin can use 2-deoxy-ATP (dATP) as an energy substrate, that it enhances contraction and relaxation with minimal effect on calcium-handling properties in vitro, and that contractile enhancement occurs with only minor elevation of cellular [dATP]. Here, we report the effect of chronically enhanced dATP concentration on cardiac function using a transgenic mouse that overexpresses the enzyme ribonucleotide reductase (TgRR), which catalyzes the rate-limiting step in de novo deoxyribonucleotide biosynthesis. Hearts from TgRR mice had elevated left ventricular systolic function compared with wild-type (WT) mice, both in vivo and in vitro, without signs of hypertrophy or altered diastolic function. Isolated cardiomyocytes from TgRR mice had enhanced contraction and relaxation, with no change in Ca(2+) transients, suggesting targeted improvement of myofilament function. TgRR hearts had normal ATP and only slightly decreased phosphocreatine levels by (31)P NMR spectroscopy, and they maintained rate responsiveness to dobutamine challenge. These data demonstrate long-term (at least 5-mo) elevation of cardiac [dATP] results in sustained elevation of basal left ventricular performance, with maintained ß-adrenergic responsiveness and energetic reserves. Combined with results from previous studies, we conclude that this occurs primarily via enhanced myofilament activation and contraction, with similar or faster ability to relax. The data are sufficiently compelling to consider elevated cardiac [dATP] as a therapeutic option to treat systolic dysfunction.

Structural and functional consequences of the cardiac troponin C L48Q Ca(2+)-sensitizing mutation.

Calcium binding to the regulatory domain of cardiac troponin C (cNTnC) causes a conformational change that exposes a hydrophobic surface to which troponin I (cTnI) binds, prompting a series of protein-protein interactions that culminate in muscle contraction. A number of cTnC variants that alter the Ca(2+) sensitivity of the thin filament have been linked to disease. Tikunova and Davis engineered a series of cNTnC mutations that altered Ca(2+) binding properties and studied the effects on the Ca(2+) sensitivity of the thin filament and contraction [Tikunova, S. B., and Davis, J. P. (2004) J. Biol. Chem. 279, 35341-35352]. One of the mutations they engineered, the L48Q variant, resulted in a pronounced increase in the cNTnC Ca(2+) binding affinity and Ca(2+) sensitivity of cardiac muscle force development. In this work, we sought structural and mechanistic explanations for the increased Ca(2+) sensitivity of contraction for the L48Q cNTnC variant, using an array of biophysical techniques. We found that the L48Q mutation enhanced binding of both Ca(2+) and cTnI to cTnC. Nuclear magnetic resonance chemical shift and relaxation data provided evidence that the cNTnC hydrophobic core is more exposed with the L48Q variant. Molecular dynamics simulations suggest that the mutation disrupts a network of crucial hydrophobic interactions so that the closed form of cNTnC is destabilized. The findings emphasize the importance of cNTnC's conformation in the regulation of contraction and suggest that mutations in cNTnC that alter myofilament Ca(2+) sensitivity can do so by modulating Ca(2+) and cTnI binding.

Ca2+ regulation of rabbit skeletal muscle thin filament sliding: role of cross-bridge number.

We investigated how strong cross-bridge number affects sliding speed of regulated Ca(2+)-activated, thin filaments. First, using in vitro motility assays, sliding speed decreased nonlinearly with reduced density of heavy meromyosin (HMM) for regulated (and unregulated) F-actin at maximal Ca(2+). Second, we varied the number of Ca(2+)-activatable troponin complexes at maximal Ca(2+) using mixtures of recombinant rabbit skeletal troponin (WT sTn) and sTn containing sTnC(D27A,D63A), a mutant deficient in Ca(2+) binding at both N-terminal, low affinity Ca(2+)-binding sites (xxsTnC-sTn). Sliding speed decreased nonlinearly as the proportion of WT sTn decreased. Speed of regulated thin filaments varied with pCa when filaments contained WT sTn but filaments containing only xxsTnC-sTn did not move. pCa(50) decreased by 0.12-0.18 when either heavy meromyosin density was reduced to approximately 60% or the fraction of Ca(2+)-activatable regulatory units was reduced to approximately 33%. Third, we exchanged mixtures of sTnC and xxsTnC into single, permeabilized fibers from rabbit psoas. As the proportion of xxsTnC increased, unloaded shortening velocity decreased nonlinearly at maximal Ca(2+). These data are consistent with unloaded filament sliding speed being limited by the number of cycling cross-bridges so that maximal speed is attained with a critical, low level of actomyosin interactions.