Cardiac troponin C (cTnC) is the regulatory protein that initiates cardiac contraction in response to Ca TnC binding Ca initiates a cascade of protein-protein interactions that begins with the opening of the N-terminal domain of cTnC, followed by cTnC binding the troponin I switch peptide (TnI). We have evaluated, through isothermal titration calorimetry and molecular-dynamics simulation, the effect of several clinically relevant mutations (A8V, L29Q, A31S, L48Q, Q50R, and C84Y) on the Ca affinity, structural dynamics, and calculated interaction strengths between cTnC and each of Ca and TnI Surprisingly the Ca affinity measured by isothermal titration calorimetry was only significantly affected by half of these mutations including L48Q, which had a 10-fold higher affinity than WT, and the Q50R and C84Y mutants, each of which had affinities 3-fold higher than wild type. This suggests that Ca affinity of the N-terminal domain of cTnC in isolation is insufficient to explain the pathogenicity of these mutations. Molecular-dynamics simulation was used to evaluate the effects of these mutations on Ca binding, structural dynamics, and TnI interaction independently. Many of the mutations had a pronounced effect on the balance between the open and closed conformations of the TnC molecule, which provides an indirect mechanism for their pathogenic properties. Our data demonstrate that the structural dynamics of the cTnC molecule are key in determining myofilament Ca sensitivity. Our data further suggest that modulation of the structural dynamics is the underlying molecular mechanism for many disease mutations that are far from the regulatory Ca-binding site of cTnC.

Cannabidiol (CBD) is the primary nonpsychotropic phytocannabinoid found in Cannabis sativa, which has been proposed to be therapeutic against many conditions, including muscle spasms. Among its putative targets are voltage-gated sodium channels (Navs), which have been implicated in many conditions. We investigated the effects of CBD on Nav1.4, the skeletal muscle Nav subtype. We explored direct effects, involving physical block of the Nav pore, as well as indirect effects, involving modulation of membrane elasticity that contributes to Nav inhibition. MD simulations revealed CBD’s localization inside the membrane and effects on bilayer properties. Nuclear magnetic resonance (NMR) confirmed these results, showing CBD localizing below membrane headgroups. To determine the functional implications of these findings, we used a gramicidin-based fluorescence assay to show that CBD alters membrane elasticity or thickness, which could alter Nav function through bilayer-mediated regulation. Site-directed mutagenesis in the vicinity of the Nav1.4 pore revealed that removing the local anesthetic binding site with F1586A reduces the block of INa by CBD. Altering the fenestrations in the bilayer-spanning domain with Nav1.4-WWWW blocked CBD access from the membrane into the Nav1.4 pore (as judged by MD). The stabilization of inactivation, however, persisted in WWWW, which we ascribe to CBD-induced changes in membrane elasticity. To investigate the potential therapeutic value of CBD against Nav1.4 channelopathies, we used a pathogenic Nav1.4 variant, P1158S, which causes myotonia and periodic paralysis. CBD reduces excitability in both wild-type and the P1158S variant. Our in vitro and in silico results suggest that CBD may have therapeutic value against Nav1.4 hyperexcitability.

The Ca2+ binding properties of the FHC-associated cardiac troponin C (cTnC) mutation L29Q were examined in isolated cTnC, troponin complexes, reconstituted thin filament preparations, and skinned cardiomyocytes. While higher Ca2+ binding affinity was apparent for the L29Q mutant in isolated cTnC, this phenomenon was not observed in the cTn complex. At the level of the thin filament in the presence of phosphomimetic TnI, L29Q cTnC further reduced the Ca2+ affinity by 27% in the steady-state measurement and increased the Ca2+ dissociation rate by 20% in the kinetic studies. Molecular dynamics simulations suggest that L29Q destabilizes the conformation of cNTnC in the presence of phosphomimetic cTnI and potentially modulates the Ca2+ sensitivity due to the changes of the opening/closing equilibrium of cNTnC. In the skinned cardiomyocyte preparation, L29Q cTnC increased Ca2+ sensitivity in a highly sarcomere length (SL)-dependent manner. The well-established reduction of Ca2+ sensitivity by phosphomimetic cTnI was diminished by 68% in the presence of the mutation and it also depressed the SL-dependent increase in myofilament Ca2+ sensitivity. This might result from its modified interaction with cTnI which altered the feedback effects of cross-bridges on the L29Q cTnC-cTnI-Tm complex. This study demonstrates that the L29Q mutation alters the contractility and the functional effects of the phosphomimetic cTnI in both thin filament and single skinned cardiomyocytes and importantly that this effect is highly sarcomere length dependent.

The zebrafish (Danio rerio) is an important organism as a model for understanding vertebrate cardiovascular development. However, little is known about adult ZF cardiac function and how contractile function changes to cope with fluctuations in ambient temperature. The goals of this study were to: 1) determine if high resolution echocardiography (HRE) in the presence of reduced cardiodepressant anesthetics could be used to accurately investigate the structural and functional properties of the ZF heart and 2) if the effect of ambient temperature changes both acutely and chronically could be determined non-invasively using HRE in vivo. Heart rate (HR) appears to be the critical factor in modifying cardiac output (CO) with ambient temperature fluctuation as it increases from 78 ± 5.9 bpm at 18°C to 162 ± 9.7 bpm at 28°C regardless of acclimation state (cold acclimated CA– 18°C; warm acclimated WA– 28°C). Stroke volume (SV) is highest when the ambient temperature matches the acclimation temperature, though this difference did not constitute a significant effect (CA 1.17 ± 0.15 μL at 18°C vs 1.06 ± 0.14 μl at 28°C; WA 1.10 ± 0.13 μL at 18°C vs 1.12 ± 0.12 μl at 28°C). The isovolumetric contraction time (IVCT) was significantly shorter in CA fish at 18°C. The CA group showed improved systolic function at 18°C in comparison to the WA group with significant increases in both ejection fraction and fractional shortening and decreases in IVCT. The decreased early peak (E) velocity and early peak velocity / atrial peak velocity (E/A) ratio in the CA group are likely associated with increased reliance on atrial contraction for ventricular filling.

Zebrafish (Danio rerio) are widely used as vertebrate model in developmental genetics and functional genomics as well as in cardiac structure-function studies. The zebrafish heart has been increasingly used as a model of human cardiac function, in part, due to the similarities in heart rate and action potential duration and morphology with respect to humans. The teleostian zebrafish is in many ways a compelling model of human cardiac function due to the clarity afforded by its ease of genetic manipulation, the wealth of developmental biological information, and inherent suitability to a variety of experimental techniques. However, in addition to the numerous advantages of the zebrafish system are also caveats related to gene duplication (resulting in paralogs not present in human or other mammals) and fundamental differences in how zebrafish hearts function. In this review, we discuss the use of zebrafish as a cardiac function model through the use of techniques such as echocardiography, optical mapping, electrocardiography, molecular investigations of excitation-contraction coupling, and their physiological implications relative to that of the human heart. While some of these techniques (e.g., echocardiography) are particularly challenging in the zebrafish because of diminutive size of the heart (~1.5 mm in diameter) critical information can be derived from these approaches and are discussed in detail in this article.

hERG channel dysfunction causes long QT syndrome and arrhythmia. Activator compounds have been of significant interest due to their therapeutic potential. We used the whole organ zebrafish heart model to demonstrate the antiarrhythmic benefit of the hERG activator, RPR260243. The activator abbreviated APD and increased refractoriness, the combined effect of which rescued induced ventricular arrhythmia. Our findings show that the targeted slowing of hERG channel deactivation and enhancement of protective currents caused by the RPR260243 activator may provide an effective antiarrhythmic approach.

Cardiac muscle thin filaments are composed of actin, tropomyosin, and troponin that change conformation in response to Ca 2+ binding, triggering muscle contraction. Human cardiac troponin C (cTnC) is the Ca 2+ -sensing component of the thin filament. It contains structural sites (III/IV) that bind both Ca 2+ and Mg 2+ and a regulatory site (II) that has been thought to bind only Ca 2+ . Binding of Ca 2+ at this site initiates a series of conformational changes that culminate in force production. However, the mechanisms that underpin the regulation of binding at site II remain unclear. Here, we have quantified the interaction between site II and Ca 2+ /Mg 2+ through isothermal titration calorimetry and thermodynamic integration simulations. Direct and competitive binding titrations with WT N-terminal cTnC and full-length cTnC indicate that physiologically relevant concentrations of both Ca 2+ /Mg 2+ interacted with the same locus. Moreover, the D67A/D73A N-terminal cTnC construct in which two coordinating residues within site II were removed was found to have significantly reduced affinity for both cations. In addition, 1 mM Mg 2+ caused a 1.4-fold lower affinity for Ca 2+ . These experiments strongly suggest that cytosolic-free Mg 2+ occupies a significant population of the available site II. Interaction of Mg 2+ with site II of cTnC likely has important functional consequences for the heart both at baseline as well as in diseased states that decrease or increase the availability of Mg 2+ , such as secondary hyperparathyroidism or ischemia, respectively.

Zebrafish, as a model for teleost fish, have two paralogous troponin C (TnC) genes that are expressed in the heart differentially in response to temperature acclimation. Upon Ca(2+) binding, TnC changes conformation and exposes a hydrophobic patch that interacts with troponin I and initiates cardiac muscle contraction. Teleost-specific TnC paralogs have not yet been functionally characterized. In this study we have modeled the structures of the paralogs using molecular dynamics simulations at 18°C and 28°C and calculated the different Ca(2+)-binding properties between the teleost cardiac (cTnC or TnC1a) and slow-skeletal (ssTnC or TnC1b) paralogs through potential-of-mean-force calculations. These values are compared with thermodynamic binding properties obtained through isothermal titration calorimetry (ITC). The modeled structures of each of the paralogs are similar at each temperature, with the exception of helix C, which flanks the Ca(2+) binding site; this region is also home to paralog-specific sequence substitutions that we predict have an influence on protein function. The short timescale of the potential-of-mean-force calculation precludes the inclusion of the conformational change on the ΔG of Ca(2+) interaction, whereas the ITC analysis includes the Ca(2+) binding and conformational change of the TnC molecule. ITC analysis has revealed that ssTnC has higher Ca(2+) affinity than cTnC for Ca(2+) overall, whereas each of the paralogs has increased affinity at 28°C compared to 18°C. Microsecond-timescale simulations have calculated that the cTnC paralog transitions from the closed to the open state more readily than the ssTnC paralog, an unfavorable transition that would decrease the ITC-derived Ca(2+) affinity while simultaneously increasing the Ca(2+) sensitivity of the myofilament. We propose that the preferential expression of cTnC at lower temperatures increases myofilament Ca(2+) sensitivity by this mechanism, despite the lower Ca(2+) affinity that we have measured by ITC.