In vitro analyses of suspected arrhythmogenic thin filament variants as a cause of sudden cardiac death in infants.

Sudden unexpected death of an infant (SUDI) is a devastating occurrence for families. To investigate the genetic pathogenesis of SUDI, we sequenced >70 genes from 191 autopsy-negative SUDI victims. Ten infants sharing a previously unknown variant in troponin I (TnI) were identified. The mutation (TNNI1 R37C+/-) is in the fetal/neonatal paralog of TnI, a gene thought to be expressed in the heart up to the first 24 months of life. Using phylogenetic analysis and molecular dynamics simulations, it was determined that arginine at residue 37 in TNNI1 may play a critical functional role, suggesting that the variant may be pathogenic. We investigated the biophysical properties of the TNNI1 R37C mutation in human reconstituted thin filaments (RTFs) using fluorometry. RTFs reconstituted with the mutant R37C TnI exhibited reduced Ca2+-binding sensitivity due to an increased Ca2+ off-rate constant. Furthermore, we generated TNNI1 R37C+/- mutants in human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs) using CRISPR-Cas9. In monolayers of hiPSC-CMs, we simultaneously monitored voltage and Ca2+ transients through optical mapping and compared them to their isogenic controls. We observed normal intrinsic beating patterns under control conditions in TNNI1 R37C+/- at stimulation frequencies of 55 beats/min (bpm), but these cells showed no restitution with increased stimulation frequency to 65 bpm and exhibited alternans at >75 bpm. The WT hiPSC-CMs did not exhibit any sign of arrhythmogenicity even at stimulation frequencies of 120 bpm. The approach used in this study provides critical physiological and mechanistic bases to investigate sarcomeric mutations in the pathogenesis of SUDI.

Investigating the Genetic Causes of Sudden Unexpected Death in Children Through Targeted Next-Generation Sequencing Analysis.

BACKGROUND: Inherited arrhythmia syndromes are responsible for a significant portion of autopsy-negative sudden unexpected death (SUD) cases, but molecular autopsy used to identify potentially causal variants is not routinely included in SUD investigations. We collaborated with a medical examiner's office to assist in finding a diagnosis for their autopsy-negative child SUD cases. METHODS AND RESULTS: 191 child SUD cases (<5 years of age) were selected for analyses. Our next generation sequencing panel incorporated 38 inherited arrhythmia syndrome candidate genes and another 33 genes not previously investigated for variants that may underlie SUDY pathophysiology. Overall, we identified 11 potentially causal disease-associated variants in 12 cases, for an overall yield of 6.3%. We also identified 31 variants of uncertain significance in 36 cases and 16 novel variants predicted to be pathogenic in silico in 15 cases. The disease-associated variants were reported to the medical examiner to notify surviving relatives and recommend clinical assessment. CONCLUSIONS: We have identified variants that may assist in the diagnosis of at least 6.3% of autopsy-negative child SUD cases and reduce risk of future SUD in surviving relatives. We recommend a cautious approach to variant interpretation. We also suggest inclusion of cardiomyopathy genes as well as other candidate SUD genes in molecular autopsy analyses.

Changes in the dynamics of the cardiac troponin C molecule explain the effects of Ca2+-sensitizing mutations.

Cardiac troponin C (cTnC) is the regulatory protein that initiates cardiac contraction in response to Ca2+ TnC binding Ca2+ 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 (TnISW). 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 Ca2+ affinity, structural dynamics, and calculated interaction strengths between cTnC and each of Ca2+ and TnISW Surprisingly the Ca2+ 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 Ca2+ 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 Ca2+ 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 Ca2+ 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 Ca2+-binding site of cTnC.

The Zebrafish Heart as a Model of Mammalian Cardiac Function.

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.

Characterization of Zebrafish Cardiac and Slow Skeletal Troponin C Paralogs by MD Simulation and ITC.

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.

Functional Divergence in Teleost Cardiac Troponin Paralogs Guides Variation in the Interaction of TnI Switch Region with TnC.

Gene duplication results in extra copies of genes that must coevolve with their interacting partners in multimeric protein complexes. The cardiac troponin (Tn) complex, containing TnC, TnI, and TnT, forms a distinct functional unit critical for the regulation of cardiac muscle contraction. In teleost fish, the function of the Tn complex is modified by the consequences of differential expression of paralogs in response to environmental thermal challenges. In this article, we focus on the interaction between TnI and TnC, coded for by genes that have independent evolutionary origins, but the co-operation of their protein products has necessitated coevolution. In this study, we characterize functional divergence of TnC and TnI paralogs, specifically the interrelated roles of regulatory subfunctionalization and structural subfunctionalization. We determined that differential paralog transcript expression in response to temperature acclimation results in three combinations of TnC and TnI in the zebrafish heart: TnC1a/TnI1.1, TnC1b/TnI1.1, and TnC1a/TnI1.5. Phylogenetic analysis of these highly conserved proteins identified functionally divergent residues in TnI and TnC. The structural and functional effect of these Tn combinations was modeled with molecular dynamics simulation to link divergent sites to changes in interaction strength. Functional divergence in TnI and TnC were not limited to the residues involved with TnC/TnI switch interaction, which emphasizes the complex nature of Tn function. Patterns in domain-specific divergent selection and interaction energies suggest that substitutions in the TnI switch region are crucial to modifying TnI/TnC function to maintain cardiac contraction with temperature changes. This integrative approach introduces Tn as a model of functional divergence that guides the coevolution of interacting proteins.

Familial hypertrophic cardiomyopathy related cardiac troponin C L29Q mutation alters length-dependent activation and functional effects of phosphomimetic troponin I*.

The Ca(2+) 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 Ca(2+) 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 Ca(2+) affinity by 27% in the steady-state measurement and increased the Ca(2+) 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 Ca(2+) sensitivity due to the changes of the opening/closing equilibrium of cNTnC. In the skinned cardiomyocyte preparation, L29Q cTnC increased Ca(2+) sensitivity in a highly sarcomere length (SL)-dependent manner. The well-established reduction of Ca(2+) sensitivity by phosphomimetic cTnI was diminished by 68% in the presence of the mutation and it also depressed the SL-dependent increase in myofilament Ca(2+) 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.

¹H, ¹³C and ¹5N resonance assignments and peptide binding site chemical shift perturbation mapping for the Escherichia coli redox enzyme chaperone DmsD.

Herein are reported the mainchain (1)H, (13)C and (15)N chemical shift assignments and amide (15)N relaxation data for Escherichia coli DmsD, a 23.3 kDa protein responsible for the correct folding and translocation of the dimethyl sulfoxide reductase enzyme complex. In addition, the observed amide chemical shift perturbations resulting from complex formation with the reductase subunit DmsA leader peptide support a model in which the 44 residue peptide makes extensive contacts across the surface of the DmsD protein.

Purification of a Tat leader peptide by co-expression with its chaperone.

We present a method for the purification of the 45 residue long leader peptide of Escherichia coli dimethyl sulfoxide reductase subunit A (DmsA(L)), a substrate of the twin arginine translocase, by co-expressing the leader peptide with its specific chaperone protein, DmsD. The peptide can be isolated from the soluble DmsA(L)/DmsD complex or conveniently from the lysate pellet fraction. The recombinant leader peptide is functionally intact as the peptide/chaperone complex can be reconstituted from purified DmsA(L) and DmsD. A construct with DmsA(L) fused to the N-terminus of DmsD (DmsA(L)-DmsD fusion) was created to further explore the properties of the leader peptide-chaperone interactions. Analytical size-exclusion chromatography in-line with multi-angle light scattering reveals that the DmsA(L)-DmsD fusion construct forms a dimer wherein each protomer binds the neighboring leader peptide. A model of this homodimeric interaction is presented.

Structural analysis of a monomeric form of the twin-arginine leader peptide binding chaperone Escherichia coli DmsD.

The redox enzyme maturation proteins play an essential role in the proofreading and membrane targeting of protein substrates to the twin-arginine translocase. Functionally, the most thoroughly characterized redox enzyme maturation protein to date is Escherichia coli DmsD (EcDmsD). Herein, we present the X-ray crystal structure of the monomeric form of the EcDmsD refined to 2.0 A resolution, with clear electron density present for each of its 204 amino acid residues. The structural data presented here complement the biochemical data previously generated regarding the function of these twin-arginine translocase leader peptide binding chaperone proteins. Docking and molecular dynamics simulation experiments were used to provide a proposed model for how this chaperone is able to recognize the leader peptide of its substrate DmsA. The interactions observed in the model are in agreement with previous biochemical data and suggest intimate interactions between the conserved twin-arginine motif of the leader peptide of E. coli DmsA and the most conserved regions on the surface of EcDmsD.

Identification of residues in DmsD for twin-arginine leader peptide binding, defined through random and bioinformatics-directed mutagenesis.

The twin-arginine translocase (Tat) system is used for the targeting and translocation of folded proteins across the cell membrane of most bacteria. Substrates of this system contain a conserved "twin-arginine" (RR) motif within their signal/leader peptide sequence. Many Tat substrates have their own system-specific chaperone called redox enzyme maturation proteins (REMPs). Here, we study the binding of DmsD, the REMP for dimethyl sulfoxide reductase in Escherichia coli, toward the RR-containing leader peptide of the catalytic subunit DmsA. We have used a multipronged approach targeted at the amino acid sequence of DmsD to define residues and regions important for recognition of the DmsA leader sequence. Residues identified through bioinformatics and THEMATICS analysis were mutated using site-directed mutagenesis. These DmsD residue variants were purified and screened with an in vitro dot-blot far-Western assay to analyze the binding to the DmsA leader sequence. Degenerative polymerase chain reaction was also used to produce a bank of random DmsD amino acid mutants, which were then screened by an in vivo bacterial two-hybrid assay. Using this hybrid method, each DmsD variant was classified into one of three groups based on their degree of interaction with the DmsA leader (none, weak, and moderate). The data from both the in vitro and in vivo analyses were then applied to a model structure of DmsD based on the crystal structure of the Salmonella typhimurium homologue. Our results illustrate the positions of important DmsD residues involved in binding the DmsA leader peptide and identify a "hot pocket" of residues important for leader binding on the structure of DmsD.