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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 individual's level of exertion and exposure to the sun while levels of water and caffeine consumed showed no correlation to one's level of hydration. We conclude that integrating a proactive, field-expedient means to monitor a unit's level of hydration can increase the combat effectiveness of units in training and combat alike. [ABSTRACT FROM AUTHOR]

Chronic hypoxia plays a central role in diverse pulmonary pathologies, but its effects on longitudinal changes in the biomechanical behavior of proximal pulmonary arteries remain poorly understood. Similarly, effects of normoxic recovery have not been well studied. Here, we report hypoxia-induced changes in composition, vasoactivity, and passive biaxial mechanics in the main branch pulmonary artery of male C57BL/6J mice exposed to 10% FiO2 for 1, 2, or 3 weeks. We observed significant changes in extracellular matrix, and consequently wall mechanics, as early as 1 week of hypoxia. While circumferential stress and stiffness returned toward normal values by 2-3 weeks of hypoxia, area fractions of cytoplasm and thin collagen fibers did not return toward normal until after 1 week of normoxic recovery. By contrast, elastic energy storage and overall distensibility remained reduced after 3 weeks of hypoxia as well as following 1 week of normoxic recovery. While smooth muscle and endothelial cell responses were attenuated under hypoxia, smooth muscle but not endothelial cell responses recovered following 1 week of subsequent normoxia. Collectively, these data suggest that homeostatic processes were unable to preserve or restore overall function, at least over a brief period of normoxic recovery. Longitudinal changes are critical in understanding large pulmonary artery remodeling under hypoxia, and its reversal, and will inform predictive models of vascular adaptation., (Copyright © 2024 by ASME.)

Adverse effects of large artery stiffening are well established in the systemic circulation; stiffening of the proximal pulmonary artery (PPA) and its sequelae are poorly understood. We combined in vivo (n = 6) with ex vivo data from cadavers (n = 8) and organ donors (n = 13), ages 18 to 89, to assess whether aging of the PPA associates with changes in distensibility, biaxial wall strain, wall thickness, vessel diameter, and wall composition. Aging exhibited significant negative associations with distensibility and cyclic biaxial strain of the PPA (p ≤ 0.05), with decreasing circumferential and axial strains of 20% and 7%, respectively, for every 10 years after 50. Distensibility associated directly with diffusion capacity of the lung (R 2  = 0.71, p = 0.03). Axial strain associated with right ventricular ejection fraction (R 2  = 0.76, p = 0.02). Aging positively associated with length of the PPA (p = 0.004) and increased luminal caliber (p = 0.05) but showed no significant association with mean wall thickness (1.19 mm, p = 0.61) and no significant differences in the proportions of mural elastin and collagen (p = 0.19) between younger (<50 years) and older (>50) ex vivo samples. We conclude that age-related stiffening of the PPA differs from that of the aorta; microstructural remodeling, rather than changes in overall geometry, may explain age-related stiffening., (© 2024 The Author(s). Physiological Reports published by Wiley Periodicals LLC on behalf of The Physiological Society and the American Physiological Society.)

Pulmonary function testing (PFT) in mice includes biomechanical assessment of lung function relevant to physiology in health and its alteration in disease, hence, it is frequently used in preclinical modeling of human lung pathologies. Despite numerous reports of PFT in mice of various ages, there is a lack of reference data for developing mice collected using consistent methods. Therefore, we profiled PFTs in male and female C57BL/6J mice from 2 to 23 wk of age, providing reference values for age- and sex-dependent changes in mouse lung biomechanics during development and young adulthood. Although males and females have similar weights at birth, females weigh significantly less than males after 5 wk of age ( P < 0.001) with largest weight gain observed between 3 and 8 wk in females and 3 and 13 wk in males, after which weight continued to increase more slowly up to 23 wk of age. Lung function parameters including static compliance and inspiratory capacity also increased rapidly between 3 and 8 wk in female and male mice, with male mice having significantly greater static compliance and inspiratory capacity than female mice ( P < 0.001). Although these parameters appear higher in males at a given age, allometric scaling showed that static compliance and inspiratory compliance were comparable between the two sexes. This suggests that differences in measurements of lung function are likely body weight-based rather than sex-based. We expect these data to facilitate future lung disease research by filling a critical knowledge gap in our field. NEW & NOTEWORTHY This study provides reference values for changes in mouse lung biomechanics from 2 to 23 wk of age. There are rapid developmental changes in lung structure and function of male and female mice between the ages of 3 and 8 wk. Male mice become noticeably heavier than female mice at or about 5 wk of age. We identified that differences in normal lung function measurements are likely weight-based, not sex-based.

Age is a major risk factor for lung disease. To understand the mechanisms underlying this association, we characterized the changing cellular, genomic, transcriptional, and epigenetic landscape of lung aging using bulk and single-cell RNAseq (scRNAseq) data. Our analysis revealed age-associated gene networks that reflected hallmarks of aging, including mitochondrial dysfunction, inflammation, and cellular senescence. Cell type deconvolution revealed age-associated changes in the cellular composition of the lung: decreased alveolar epithelial cells and increased fibroblasts and endothelial cells. In the alveolar microenvironment, aging is characterized by decreased AT2B cells and reduced surfactant production, a finding that was validated by scRNAseq and IHC. We showed that a previously reported senescence signature, SenMayo, captures cells expressing canonical senescence markers. SenMayo signature also identified cell-type specific senescence-associated co-expression modules that have distinct molecular functions, including ECM regulation, cell signaling, and damage response pathways. Analysis of somatic mutations showed that burden was highest in lymphocytes and endothelial cells and was associated with high expression of senescence signature. Finally, aging and senescence gene expression modules were associated with differentially methylated regions, with inflammatory markers such as IL1B , IL6R , and TNF being significantly regulated with age. Our findings provide new insights into the mechanisms underlying lung aging and may have implications for the development of interventions to prevent or treat age-related lung diseases., Competing Interests: Conflict of Interest NK is a scientific founder at Thyron, served as a consultant to Biogen Idec, Boehringer Ingelheim, Third Rock, Pliant, Samumed, NuMedii, Theravance, LifeMax, Three Lake Partners, Optikira, Astra Zeneca, RohBar, Veracyte, Augmanity, CSL Behring, Galapagos, Fibrogen, and Thyron over the last 3 years, reports Equity in Pliant and Thyron, and grants from Veracyte, Boehringer Ingelheim, BMS and non-financial support from MiRagen and Astra Zeneca. LN is the founder, President and CEO of Humacyte Global Inc, a publicly traded regenerative medicine company.

Purpose: Tracheostomy is a necessary procedure required for prolonged mechanical ventilation in long-term acute care hospitals (LTACH). Many factors influence successful decannulation, or tracheostomy removal, and it is unclear what factors are essential for determining decannulation. The purpose of this study was to determine retrospective performance of single prognostic variables for successful decannulation, like peak expiratory flow measurement, overnight oximetry testing, and blood gas analysis., Methods: A retrospective analysis of a three-year period to investigate the association between peak flow (PF) measurements ≥160 L/min, successful overnight oximetry (ONO), sex, and decannulation success. Average PF measurements, arterial blood gas (ABG), days on mechanical ventilation, LTACH length of stay (LOS), and age were also investigated., Results: We examined the records of 135 patients, 127 of which were successfully decannulated. PF measurements ≥160 L/min (p=0.16), sex (p<0.05) and passing ONO (p<0.05) were significantly different between successfully and unsuccessfully decannulated patients; mean ABG (pH, pCO2, pO2), mechanical ventilation days, LOS, and age were not significantly different (p>0.05)., Conclusions: These results suggest no single prognostic variable can predict decannulation outcomes. Rather, clinical judgment of experienced medical professionals appears sufficient to achieve a 94% decannulation success rate. Additional investigation is required to determine what metrics are necessary, or if clinical judgment alone can predict decannulation success.

Prolonged time on mechanical ventilation is associated with multiple consequences for both the patient and medical facility. Based on anecdotal evidence that sustained elevation of beta-natriuretic protein (BNP) during a patient's stay in a long-term acute care hospital (LTACH) was associated with failure to wean from prolonged mechanical ventilation, we investigated if there is an association between a decrease in BNP levels during one's stay and successful weaning from prolonged mechanical ventilation. We performed a retrospective study of 66 patient records revealing no correlation between lowering BNP levels and probability of liberating a patient from prolonged mechanical ventilation in an LTACH environment where the probability of liberation from mechanical ventilation is high (> 85%). BNP measurements by itself does not appear to be a helpful tool in the likelihood of liberation from mechanical ventilation AUC = 0.61 (CI: 0.48-0.72). In an LTACH setting with high success rates of liberation from mechanical ventilation, it does not appear to be necessary to trend BNP measurements in attempts to liberate patients from prolonged mechanical ventilation.

Aims: Studies have linked severe hyperoxia, or prolonged exposure to very high oxygen levels, with worse clinical outcomes. This study investigated the role of epidermal growth factor receptor (EGFR) in hyperoxia-induced lung injury at very high oxygen levels (>95%)., Results: Effects of severe hyperoxia (100% oxygen) were studied in mice with genetically inhibited EGFR and wild-type littermates. Despite the established role of EGFR in lung repair, EGFR inhibition led to improved survival and reduced acute lung injury, which prompted an investigation into this protective mechanism. Endothelial EGFR genetic knockout did not confer protection. EGFR inhibition led to decreased levels of cleaved caspase-3 and poly (ADP-ribosyl) polymerase (PARP) and decreased terminal dUTP nick end labeling- (TUNEL-) positive staining in alveolar epithelial cells and reduced ERK activation, which suggested reduced apoptosis in vivo . EGFR inhibition decreased hyperoxia (95%)-induced apoptosis and ERK in murine alveolar epithelial cells in vitro , and CRISPR-mediated EGFR deletion reduced hyperoxia-induced apoptosis and ERK in human alveolar epithelial cells in vitro . Innovation . This work defines a protective role of EGFR inhibition to decrease apoptosis in lung injury induced by 100% oxygen. This further characterizes the complex role of EGFR in acute lung injury and outlines a novel hyperoxia-induced cell death pathway that warrants further study., Conclusion: In conditions of severe hyperoxia (>95% for >24 h), EGFR inhibition led to improved survival, decreased lung injury, and reduced cell death. These findings further elucidate the complex role of EGFR in acute lung injury., Competing Interests: The authors declare that there is no conflict of interest regarding the publication of this article., (Copyright © 2022 Zachary M. Harris et al.)

Competent elastic fibers are critical to the function of the lung and right circulation. Murine models of elastopathies can aid in understanding the functional roles of the elastin and elastin-associated glycoproteins that constitute elastic fibers. Here, we quantify together lung and pulmonary arterial structure, function, and mechanics with right heart function in a mouse model deficient in the elastin-associated glycoprotein fibulin-5. Differences emerged as a function of genotype, sex, and arterial region. Specifically, functional studies revealed increased lung compliance in fibulin-5 deficiency consistent with a histologically observed increased alveolar disruption. Biaxial mechanical tests revealed that the primary branch pulmonary arteries exhibit decreased elastic energy storage capacity and wall stress despite only modest differences in circumferential and axial material stiffness in the fibulin-5 deficient mice. Histological quantifications confirm a lower elastic fiber content in the fibulin-5 deficient pulmonary arteries, with fragmented elastic laminae in the outer part of the wall - likely the reason for reduced energy storage. Ultrasound measurements confirm sex differences in compromised right ventricular function in the fibulin-5 deficient mice. These results reveal compromised right heart function, but opposite effects of elastic fiber dysfunction on the lung parenchyma (significantly increased compliance) and pulmonary arteries (trend toward decreased distensibility), and call for further probing of ventilation-perfusion relationships in pulmonary pathologies. Amongst many other models, fibulin-5 deficient mice can contribute to our understanding of the complex roles of elastin in pulmonary health and disease., (Copyright © 2022 by ASME.)

Hypoxia adversely affects the pulmonary circulation of mammals, including vasoconstriction leading to elevated pulmonary arterial pressures. The clinical importance of changes in the structure and function of the large, elastic pulmonary arteries is gaining increased attention, particularly regarding impact in multiple chronic cardiopulmonary conditions. We establish a multi-disciplinary workflow to understand better transcriptional, microstructural, and functional changes of the pulmonary artery in response to sustained hypoxia and how these changes inter-relate. We exposed adult male C57BL/6J mice to normoxic or hypoxic (FiO 2 10%) conditions. Excised pulmonary arteries were profiled transcriptionally using single cell RNA sequencing, imaged with multiphoton microscopy to determine microstructural features under in vivo relevant multiaxial loading, and phenotyped biomechanically to quantify associated changes in material stiffness and vasoactive capacity. Pulmonary arteries of hypoxic mice exhibited an increased material stiffness that was likely due to collagen remodeling rather than excessive deposition (fibrosis), a change in smooth muscle cell phenotype reflected by decreased contractility and altered orientation aligning these cells in the same direction as the remodeled collagen fibers, endothelial proliferation likely representing endothelial-to-mesenchymal transitioning, and a network of cell-type specific transcriptomic changes that drove these changes. These many changes resulted in a system-level increase in pulmonary arterial pulse wave velocity, which may drive a positive feedback loop exacerbating all changes. These findings demonstrate the power of a multi-scale genetic-functional assay. They also highlight the need for systems-level analyses to determine which of the many changes are clinically significant and may be potential therapeutic targets., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Manning, Ramachandra, Schupp, Cavinato, Raredon, Bärnthaler, Cosme, Singh, Tellides, Kaminski and Humphrey.)

Background: Cellular diversity of the lung endothelium has not been systematically characterized in humans. We provide a reference atlas of human lung endothelial cells (ECs) to facilitate a better understanding of the phenotypic diversity and composition of cells comprising the lung endothelium., Methods: We reprocessed human control single-cell RNA sequencing (scRNAseq) data from 6 datasets. EC populations were characterized through iterative clustering with subsequent differential expression analysis. Marker genes were validated by fluorescent microscopy and in situ hybridization. scRNAseq of primary lung ECs cultured in vitro was performed. The signaling network between different lung cell types was studied. For cross-species analysis or disease relevance, we applied the same methods to scRNAseq data obtained from mouse lungs or from human lungs with pulmonary hypertension., Results: Six lung scRNAseq datasets were reanalyzed and annotated to identify >15 000 vascular EC cells from 73 individuals. Differential expression analysis of EC revealed signatures corresponding to endothelial lineage, including panendothelial, panvascular, and subpopulation-specific marker gene sets. Beyond the broad cellular categories of lymphatic, capillary, arterial, and venous ECs, we found previously indistinguishable subpopulations; among venous EC, we identified 2 previously indistinguishable populations: pulmonary-venous ECs (COL15A1 neg ) localized to the lung parenchyma and systemic-venous ECs (COL15A1 pos ) localized to the airways and the visceral pleura; among capillary ECs, we confirmed their subclassification into recently discovered aerocytes characterized by EDNRB , SOSTDC1 , and TBX2 and general capillary EC. We confirmed that all 6 endothelial cell types, including the systemic-venous ECs and aerocytes, are present in mice and identified endothelial marker genes conserved in humans and mice. Ligand-receptor connectome analysis revealed important homeostatic crosstalk of EC with other lung resident cell types. scRNAseq of commercially available primary lung ECs demonstrated a loss of their native lung phenotype in culture. scRNAseq revealed that endothelial diversity is maintained in pulmonary hypertension. Our article is accompanied by an online data mining tool (www.LungEndothelialCellAtlas.com)., Conclusions: Our integrated analysis provides a comprehensive and well-crafted reference atlas of ECs in the normal lung and confirms and describes in detail previously unrecognized endothelial populations across a large number of humans and mice.

Introduction: Personal protective equipment shortages require the reuse of N95 respirators. We sought the necessary conditions for ozone to disinfect N95 respirators for reuse and the effects of multiple cycles of exposure., Methods: Portions of 3M 1870 N95 respirators were exposed to ozone at 400 ppm with 80% humidity for 2 hours to determine effectiveness of ozone on killing Pseudomonas aeruginosa . Entire 3M 1870 N95 respirators were exposed to five cycles of 400 ppm with 80% or higher humidity for 2 hours then evaluated for ozone's effects on airflow resistance, filtration efficiency, strap strength and quantitative fit., Results: Ozone exposure disinfected 3M 1870 N95 respirators heavily inoculated with P. aeruginosa . Ozone exposure did not negatively affect the airflow resistance, filtration efficiency, strap strength or fit of the 3M 1870 N95 respirator., Discussion: These results suggest that ozone is a feasible strategy to disinfect N95 respirators for reuse during this and future pandemics., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2021. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Sarcoidosis is an unpredictable granulomatous disease in which African Americans disproportionately experience aggressive phenotypes. Mitochondrial DNA (mtDNA) released by cells in response to various stressors contributes to tissue remodelling and inflammation. While extracellular mtDNA has emerged as a biomarker in multiple diseases, its relevance to sarcoidosis remains unknown. We aimed to define an association between extracellular mtDNA and clinical features of sarcoidosis.Extracellular mtDNA concentrations were measured using quantitative PCR for the human MT-ATP6 gene in bronchoalveolar (BAL) and plasma samples from healthy controls and patients with sarcoidosis from The Yale Lung Repository; associations between MT-ATP6 concentrations and Scadding stage, extrapulmonary disease and demographics were sought. Results were validated in the Genomic Research in Alpha-1 Antitrypsin Deficiency and Sarcoidosis cohort.Relative to controls, MT-ATP6 concentrations in sarcoidosis subjects were robustly elevated in the BAL fluid and plasma, particularly in the plasma of patients with extrapulmonary disease. Relative to Caucasians, African Americans displayed excessive MT-ATP6 concentrations in the BAL fluid and plasma, for which the latter compartment correlated with significantly higher odds of extrapulmonary disease.Enrichments in extracellular mtDNA in sarcoidosis are associated with extrapulmonary disease and African American descent. Further study into the mechanistic basis of these clinical findings may lead to novel pathophysiologic and therapeutic insights., Competing Interests: Conflict of interest: C. Ryu has nothing to disclose. Conflict of interest: C. Brandsdorfer has nothing to disclose. Conflict of interest: T. Adams has nothing to disclose. Conflict of interest: B. Hu has nothing to disclose. Conflict of interest: D.W. Kelleher has nothing to disclose. Conflict of interest: M. Yaggi has nothing to disclose. Conflict of interest: E.P. Manning has nothing to disclose. Conflict of interest: A. Walia has nothing to disclose. Conflict of interest: B. Reeves has nothing to disclose. Conflict of interest: H. Pan has nothing to disclose. Conflict of interest: J. Winkler has nothing to disclose. Conflict of interest: M. Minasyan has nothing to disclose. Conflict of interest: C.S. Dela Cruz has nothing to disclose. Conflict of interest: N. Kaminski has nothing to disclose. Conflict of interest: M. Gulati has nothing to disclose. Conflict of interest: E.L. Herzog has nothing to disclose., (Copyright ©ERS 2019.)

Background: Hyperimmunoglobulin E syndrome (HIES) is a rare primary immunodeficiency disorder defined by high serum immunoglobulin E titers that is associated with recurrent respiratory infections, formation of pneumoatoceles, recurrent skin abscesses, and characteristic dental and skeletal abnormalities., Case Presentation: We report a case of a 56-year-old male with a history of HIES, cavitary mycetomas, and allergic bronchopulmonary aspergillosis who presented with recurrent massive hemoptysis. Bronchial artery angiography and bronchoscopy failed to identify active hemorrhage, and two embolizations of the bronchial artery did not resolve the bleeding. Subsequently, selective pulmonary artery angiography was conducted that demonstrated a subsegmental pulmonary artery branch pseudoaneurysm with extravasation into an adjacent lung cavity. This was treated successfully with transcatheter embolization., Conclusions: To our knowledge, this is the first case reported of pulmonary artery pseudoaneurysm in HIES in the medical literature. Pulmonary artery pseudoaneurysm should be considered in the differential diagnosis in patients with HIES and massive hemoptysis.

Medical students are expected to perform common procedures such as suturing on patients during their third-year clerkships. However, these experiences are often viewed by medical students as stressors rather than opportunities for learning. The source of this stress is the lack of instruction on common procedures prior to being asked to observe or perform the procedure on a patient. First-time exposures to procedures in stressful environments may result in decreased confidence in medical students and decrease the frequency with which they perform these procedures in the future. The authors sought to change this paradigm by: (1) introducing a suturing module to first-year medical students in the context of the anatomy dissection laboratory and (2) measuring its effects on student attitudes and behavior over the course of their third-year clerkships when they encounter patients. The authors found that early and prolonged introduction to suturing was associated with increased student confidence relative to suturing a patient. Participation in the suturing module was associated with increased student confidence in identifying suturing instruments (P < 0.001) and suturing patients (P = 0.013). Further it positively affected their behavior as demonstrated by increased performance of suturing events from students exposed to the suturing module. (P < 0.001) This study demonstrates that early and prolonged opportunities to practice a procedural skill in a low-stress environment increases student confidence during patient interactions and alters student behavior., (© 2018 American Association of Anatomists.)

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.

Phytobezoars are a rare cause of small bowel obstruction. Such cases are most commonly associated with previous abdominal surgery or poor dentition or psychiatric conditions. A 40 year old man with a virgin abdomen and excellent dentition and no underlying psychiatric condition presented with an acute abdomen. CT scan revealed a transition point between dilated proximal loops of small bowel and collapsed distal loops. Exploratory laparotomy revealed a phytobezoar unable to be milked into the cecum and an enterectomy with primary anastamosis was performed without complication. A detailed history revealing several less common predisposing factors for phytobezoars should increase clinical suspicion of a phytobezoarinduced small bowel obstruction in the setting of an acute abdomen. Vigilance in presentations of an acute abdomen improves the usefulness of medical imaging, such as a CT, to detect phytobezoars. Understanding mechanisms of phytobezoar formation helps guide management and may prevent surgery.

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., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

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.

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.

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.