Edward P. Manning

A model of calcium-activation of the cardiac thin filament

The cardiac thin filament regulates actomyosin interactions through calcium-dependent alterations in the dynamics of cardiac troponin and tropomyosin. Over the past several decades, many details of the structure and function of the cardiac thin filament and its components have been elucidated. We propose a dynamic, complete model of the thin filament that encompasses known structures of cardiac troponin, tropomyosin, and actin and show that it is able to capture key experimental findings. By performing molecular dynamics simulations under two conditions, one with calcium bound and the other without calcium bound to site II of cardiac troponin C (cTnC), we found that subtle changes in structure and protein contacts within cardiac troponin resulted in sweeping changes throughout the complex that alter tropomyosin (Tm) dynamics and cardiac troponin--actin interactions. Significant calcium-dependent changes in dynamics occur throughout the cardiac troponin complex, resulting from the combination of the following: structural changes in the N-lobe of cTnC at and adjacent to sites I and II and the link between them; secondary structural changes of the cardiac troponin I (cTnI) switch peptide, of the mobile domain, and in the vicinity of residue 25 of the N-terminus; secondary structural changes in the cardiac troponin T (cTnT) linker and Tm-binding regions; and small changes in cTnC-cTnI and cTnT-Tm contacts. As a result of these changes, we observe large changes in the dynamics of the following regions: the N-lobe of cTnC, the mobile domain of cTnI, the I-T arm, the cTnT linker, and overlapping Tm. Our model demonstrates a comprehensive mechanism for calcium activation of the cardiac thin filament consistent with previous, independent experimental findings. This model provides a valuable tool for research into the normal physiology of cardiac myofilaments and a template for studying cardiac thin filament mutations that cause human cardiomyopathies.

Correlation of molecular and functional effects of mutations in cardiac troponin T linked to familial hypertrophic cardiomyopathy: an integrative in silico/in vitro approach.

Background: Cardiomyopathy-causing mutations in the N-terminal domain of cTnT disrupt thin filament function. Results: Amino acid substitutions at cTnT residue 92 alter motility, whereas computational analysis reveals significant changes in structural forces. Conclusion: The observed changes in thin filament function can be correlated to changes in bending forces. Significance: Coupling computation to in vitro measurements extends our understanding of disease mechanisms. Nearly 70% of all of the known cTnT mutations that cause familial hypertrophic cardiomyopathy fall within the TNT1 region that is critical to cTn-Tm binding. The high resolution structure of this domain has not been determined, and this lack of information has hindered structure-function analysis. In the current study, a coupled computational experimental approach was employed to correlate changes in cTnT dynamics to basic function using the regulated in vitro motility assay (R-IVM). An in silico approach to calculate forces in terms of a bending coordinate was used to precisely identify decreases in bending forces at residues 105 and 106 within the proposed cTnT “hinge” region. Significant functional changes were observed in multiple functional properties, including a decrease in the cooperativity of calcium activation, the calcium sensitivity of sliding speed, and maximum sliding speed. Correlation of the computational and experimental findings revealed an association between TNT1 flexibility and the cooperativity of thin filament calcium activation where an increase in flexibility led to a decrease in cooperativity. Further analysis of the primary sequence of the TNT1 region revealed a unique pattern of conserved charged TNT1 residues altered by the R92W and R92L mutations and may represent the underlying “structure” modulating this central functional domain. These data provide a framework for further integrated in silico/in vitro approaches that may be extended into a high-throughput predictive screen to overcome the current structural limitations in linking molecular phenotype to genotype in thin filament cardiomyopathies.

Molecular Effects of Familial Hypertrophic Cardiomyopathy-Related Mutations in the TNT1 Domain of cTnT

Familial hypertrophic cardiomyopathy (FHC) is one of the most common genetic causes of heart disease. Approximately 15% of FHC-related mutations are found in cTnT [cardiac troponin (cTn) T]. Most of the cTnT FHC-related mutations are in or flanking the N-tail TNT1 domain that directly interacts with overlapping tropomyosin (Tm). We investigate two sets of cTnT mutations at opposite ends of TNT1, mutations in residue 92 in the Tm-Tm overlap region of TNT1 and mutations in residues 160 and 163 in the C-terminal portion of TNT1 adjacent to the cTnT H1-H2 linker. Though all the mutations are located within TNT1, they have widely different phenotypes clinically and biophysically. Using a complete atomistic model of the cTn-Tm complex, we identify mechanisms by which the effects of TNT1 mutations propagate to the cTn core and site II of cTnC, where calcium binding and dissociation occurs. We find that mutations in TNT1 alter the flexibility of TNT1, which is inversely proportional to the cooperativity of calcium activation of the thin filament. Further, we identify a pathway of propagation of structural and dynamic changes from TNT1 to site II of cTnC, including TNT1, cTnT linker, I-T arm, regulatory domain of cTnI, the D-E linker of cTnC, and site II cTnC. Mutationally induced changes at site II of cTnC alter calcium coordination that corresponds to biophysical measurements of calcium sensitivity. Finally, we compare this pathway of mutational propagation with that of the calcium activation of the thin filament and find that they are identical but opposite in direction.

Computational Characterization of Mutations in Cardiac Troponin T Known to Cause Familial Hypertrophic Cardiomyopathy.

Cardiac Troponin T (cTnT) is a central modulator of thin filament regulation of myofilament activation. The lack of structural data for the TNT1 tail domain, a proposed α-helical region, makes the functional implications of the FHC mutations difficult to determine. Studies have suggested that flexibility of TNT1 is important in normal protein-protein interactions within the thin filament. Our groups have previously shown through Molecular Dynamics (MD) simulations that some FHC mutations, Arg92Leu(R92L) and Arg92Trp(R92W), result in increased flexibility at a critical hinge region 12 residues distant from the mutation. To explain this distant effect and its implications for FHC mutations, we characterized the dynamics of wild type and mutational segments of cTnT using MD. Our data shows an opening of the helix between residues 105-110 in mutants. Consequently, the dihedral angles of these residues correspond to non-α-helical regions on Ramachandran plots. We hypothesize the removal of a charged residue decreases electrostatic repulsion between the point mutation and surrounding residues resulting in local helical compaction. Constrained ends of the helix and localized compaction results in expansion within the nearest non-polar helical turn from the mutation site, residues 105-109.

Spatially selective immobilization of ferritin using solid–solution interfacial interactions

Spatially selective immobilization of ferritin, an iron-storage protein, on a silicon surface using a Si–SiO2 template is demonstrated. The material-specific immobilization of the protein is achieved by controlling physical forces at the solution–solid interface between a ferritin solution and the template. The electrostatic interaction and hydrophobic interaction between ferritin molecules and the two kinds of materials of the template can be tuned to achieve adsorption of ferritin only on Si. This method was used to generate spatially confined assemblies of ferritin on the nanoscale and on the micron scale.

Dehydration in extreme temperatures while conducting stability and support operations in a combat zone.

This analysis reports the effects of extreme temperatures (temperatures exceeding 100 degrees F) on the hydration of infantry-men conducting stability and support operations during phase IV Operation Iraqi Freedom in the months of June and July 2003 in An Nasiriyah, Iraq. Fifty-three infantrymen were evaluated for signs of dehydration after completing questionnaires regarding their activities during the previous 24 hours. We conducted an analysis comparing these activities and their state of hydration. The most significant factors contributing to dehydration in extreme environments proved to be the individuals level of exertion and exposure to the sun while levels of water and caffeine consumed showed no correlation to ones level of hydration. We conclude that integrating a proactive, field-expedient means to monitor a units level of hydration can increase the combat effectiveness of units in training and combat alike.

Central Nervous System Oxygen Toxicity and Hyperbaric Oxygen Seizures.

INTRODUCTION The use of hyperbaric oxygen (O2) as a therapeutic agent carries with it the risk of central nervous system (CNS) O2 toxicity. METHODS To further the understanding of this risk and the nature of its molecular mechanism, a review was conducted on the literature from various fields. RESULTS Numerous physiological changes are produced by increased partial pressures of oxygen (Po2), which may ultimately result in CNS O2 toxicity. The human body has several equilibrated safeguards that minimize effects of reactive species on neural networks, believed to play a primary role in CNS O2 toxicity. Increased partial pressure of oxygen (Po2) appears to saturate protective enzymes and unfavorably shift protective reactions in the direction of neural network overstimulation. Certain regions of the CNS appear more susceptible than others to these effects. Failure to decrease the elevated Po2 can result in a tonic-clonic seizure and death. Randomized, controlled studies in human populations would require a multicenter trial over a long period of time with numerous endpoints used to identify O2 toxicity. CONCLUSIONS The mounting scientific evidence and apparent increase in the number of hyperbaric O2 treatments demonstrate a need for further study in the near future.