Differential sensitivity to longitudinal and transverse stretch mediates transcriptional responses in mouse neonatal ventricular myocytes.

To identify how cardiomyocyte mechanosensitive signaling pathways are regulated by anisotropic stretch, micropatterned mouse neonatal cardiomyocytes were stretched primarily longitudinally or transversely to the myofiber axis. Four hours of static, longitudinal stretch induced differential expression of 557 genes, compared with 30 induced by transverse stretch, measured using RNA-seq. A logic-based ordinary differential equation model of the cardiac myocyte mechanosignaling network, extended to include the transcriptional regulation and expression of 784 genes, correctly predicted measured expression changes due to anisotropic stretch with 69% accuracy. The model also predicted published transcriptional responses to mechanical load in vitro or in vivo with 63-91% accuracy. The observed differences between transverse and longitudinal stretch responses were not explained by differential activation of specific pathways but rather by an approximately twofold greater sensitivity to longitudinal stretch than transverse stretch. In vitro experiments confirmed model predictions that stretch-induced gene expression is more sensitive to angiotensin II and endothelin-1, via RhoA and MAP kinases, than to the three membrane ion channels upstream of calcium signaling in the network. Quantitative cardiomyocyte gene expression differs substantially with the axis of maximum principal stretch relative to the myofilament axis, but this difference is due primarily to differences in stretch sensitivity rather than to selective activation of mechanosignaling pathways.NEW & NOTEWORTHY Anisotropic stretch applied to micropatterned neonatal mouse ventricular myocytes induced markedly greater acute transcriptional responses when the major axis of stretch was parallel to the myofilament axis than when it was transverse. Analysis with a novel quantitative network model of mechanoregulated cardiomyocyte gene expression suggests that this difference is explained by higher cell sensitivity to longitudinal loading than transverse loading than by the activation of differential signaling pathways.

Applying a Military Teleophthalmology Mobile App in a Noncombat Emergent Care Setting.

INTRODUCTION: Teleophthalmology has a natural role in the military due to the inherent organization of its medical system, which provides care to patients in remote locations around the world. Improving access to ophthalmic care enhances force readiness because ocular trauma and disease can cause vision impairment or blindness and can occur anywhere service members are located. Recently, a secure, Health Insurance Portability and Accountability Act-compliant mobile phone application (app) for teleophthalmology called Forward Operating Base Expert Telemedicine Resource Utilizing Mobile Application for Trauma (FOXTROT) was beta tested in Afghanistan and demonstrated that this solution can improve and extend ophthalmic care in a deployed environment. There are few civilian or military teleophthalmology solutions for ocular trauma and disease in an urgent or emergent ophthalmic care setting. Civilian teleophthalmology solutions have largely been developed for disease-specific models of care. In this work, we address this gap by testing the FOXTROT app in a non-deployed, emergent care setting. MATERIALS AND METHODS: We evaluated the use of the teleophthalmology mobile phone app (FOXTROT) in a non-deployed military setting at the Malcolm Grow Medical Clinics and Surgery Center at Joint Base Andrews in Maryland. Consults from the emergent care center were placed by providers using the app, and the on-call ophthalmologist responded with treatment and management recommendations. The primary outcomes were response within the requested time, visual acuity tested in both eyes, agreement between the teleophthalmology and the final diagnosis, and the number of communication or technical errors that prevented the completion of consults. The secondary outcomes were average response time and the number of consults uploaded to the medical record. RESULTS: From October 2020 to January 2022, 109 consults were received. Ten consults had communication or technical errors that prevented the completion of consults within the app and were excluded from the analysis of completed consults. Of the 99 completed consults, responses were given within the requested time in 95 (96.0%), with the average response time in 11 minutes 48 seconds (95% confidence interval, 8 minutes 57 seconds to 14 minutes 41 seconds). Visual acuity was tested in both eyes in 56 (56.6%). There was agreement between the teleophthalmology diagnosis and the final diagnosisin 40 of 50 (80.0%) consults with both a teleophthalmology and final diagnosis. Ninety-eight (99.0%) consults were uploaded to the patient's medical record. CONCLUSIONS: Beta testing of a teleophthalmology mobile phone app (FOXTROT) in a noncombat emergent care setting demonstrated that this solution can extend ophthalmic care in this environment at a military treatment facility. However, improvements in the reliability of the platform are needed in future developments to reduce communication and technical errors.

Three-dimensional transistor arrays for intra- and inter-cellular recording.

Electrical impulse generation and its conduction within cells or cellular networks are the cornerstone of electrophysiology. However, the advancement of the field is limited by sensing accuracy and the scalability of current recording technologies. Here we describe a scalable platform that enables accurate recording of transmembrane potentials in electrogenic cells. The platform employs a three-dimensional high-performance field-effect transistor array for minimally invasive cellular interfacing that produces faithful recordings, as validated by the gold standard patch clamp. Leveraging the high spatial and temporal resolutions of the field-effect transistors, we measured the intracellular signal conduction velocity of a cardiomyocyte to be 0.182 m s-1, which is about five times the intercellular velocity. We also demonstrate intracellular recordings in cardiac muscle tissue constructs and reveal the signal conduction paths. This platform could provide new capabilities in probing the electrical behaviours of single cells and cellular networks, which carries broad implications for understanding cellular physiology, pathology and cell-cell interactions.

Distinct time courses and mechanics of right ventricular hypertrophy and diastolic stiffening in a male rat model of pulmonary arterial hypertension.

Although pulmonary arterial hypertension (PAH) leads to right ventricle (RV) hypertrophy and structural remodeling, the relative contributions of changes in myocardial geometric and mechanical properties to systolic and diastolic chamber dysfunction and their time courses remain unknown. Using measurements of RV hemodynamic and morphological changes over 10 wk in a male rat model of PAH and a mathematical model of RV mechanics, we discriminated the contributions of RV geometric remodeling and alterations of myocardial material properties to changes in systolic and diastolic chamber function. Significant and rapid RV hypertrophic wall thickening was sufficient to stabilize ejection fraction in response to increased pulmonary arterial pressure by week 4 without significant changes in systolic myofilament activation. After week 4, RV end-diastolic pressure increased significantly with no corresponding changes in end-diastolic volume. Significant RV diastolic chamber stiffening by week 5 was not explained by RV hypertrophy. Instead, model analysis showed that the increases in RV end-diastolic chamber stiffness were entirely attributable to increased resting myocardial material stiffness that was not associated with significant myocardial fibrosis or changes in myocardial collagen content or type. These findings suggest that whereas systolic volume in this model of RV pressure overload is stabilized by early RV hypertrophy, diastolic dilation is prevented by subsequent resting myocardial stiffening.NEW & NOTEWORTHY Using a novel combination of hemodynamic and morphological measurements over 10 wk in a male rat model of PAH and a mathematical model of RV mechanics, we found that compensated systolic function was almost entirely explained by RV hypertrophy, but subsequently altered RV end-diastolic mechanics were primarily explained by passive myocardial stiffening that was not associated with significant collagen extracellular matrix accumulation.

Substrate Stiffness and Stretch Regulate Profibrotic Mechanosignaling in Pulmonary Arterial Adventitial Fibroblasts.

Pulmonary arterial adventitial fibroblasts (PAAFs) are important regulators of fibrotic vascular remodeling during the progression of pulmonary arterial hypertension (PAH), a disease that currently has no effective anti-fibrotic treatments. We conducted in-vitro experiments in PAAFs cultured on hydrogels attached to custom-made equibiaxial stretchers at 10% stretch and substrate stiffnesses representing the mechanical conditions of mild and severe stages of PAH. The expression of collagens α(1)I and α(1)III and elastin messenger RNAs (Col1a1, Col3a1, Eln) were upregulated by increased stretch and substrate stiffness, while lysyl oxidase-like 1 and α-smooth muscle actin messenger RNAs (Loxl1, Acta2) were only significantly upregulated when the cells were grown on matrices with an elevated stiffness representative of mild PAH but not on a stiffness representative of severe PAH. Fibronectin messenger RNA (Fn1) levels were significantly induced by increased substrate stiffness and transiently upregulated by stretch at 4 h, but was not significantly altered by stretch at 24 h. We modified our published computational network model of the signaling pathways that regulate profibrotic gene expression in PAAFs to allow for differential regulation of mechanically-sensitive nodes by stretch and stiffness. When the model was modified so that stiffness activated integrin ß3, the Macrophage Stimulating 1 or 2 (MST1\2) kinases, angiotensin II (Ang II), transforming growth factor-ß (TGF-ß), and syndecan-4, and stretch-regulated integrin ß3, MST1\2, Ang II, and the transient receptor potential (TRP) channel, the model correctly predicted the upregulation of all six genes by increased stiffness and the observed responses to stretch in five out of six genes, although it could not replicate the non-monotonic effects of stiffness on Loxl1 and Acta2 expression. Blocking Ang II Receptor Type 1 (AT1R) with losartan in-vitro uncovered an interaction between the effects of stretch and stiffness and angiotensin-independent activation of Fn1 expression by stretch in PAAFs grown on 3-kPa matrices. This novel combination of in-vitro and in-silico models of PAAF profibrotic cell signaling in response to altered mechanical conditions may help identify regulators of vascular adventitial remodeling due to changes in stretch and matrix stiffness that occur during the progression of PAH in-vivo.

Methods and sensors for functional genomic studies of cell-cycle transitions in single cells.

Much of our understanding of the regulatory mechanisms governing the cell cycle in mammals has relied heavily on methods that measure the aggregate state of a population of cells. While instrumental in shaping our current understanding of cell proliferation, these approaches mask the genetic signatures of rare subpopulations such as quiescent (G0) and very slowly dividing (SD) cells. Results described in this study and those of others using single-cell analysis reveal that even in clonally derived immortalized cancer cells, ∼1-5% of cells can exhibit G0 and SD phenotypes. Therefore to enable the study of these rare cell phenotypes we established an integrated molecular, computational, and imaging approach to track, isolate, and genetically perturb single cells as they proliferate. A genetically encoded cell-cycle reporter (K67p-FUCCI) was used to track single cells as they traversed the cell cycle. A set of R-scripts were written to quantify K67p-FUCCI over time. To enable the further study G0 and SD phenotypes, we retrofitted a live cell imaging system with a micromanipulator to enable single-cell targeting for functional validation studies. Single-cell analysis revealed HT1080 and MCF7 cells had a doubling time of ∼24 and ∼48 h, respectively, with high duration variability in G1 and G2 phases. Direct single-cell microinjection of mRNA encoding (GFP) achieves detectable GFP fluorescence within ∼5 h in both cell types. These findings coupled with the possibility of targeting several hundreds of single cells improves throughput and sensitivity over conventional methods to study rare cell subpopulations.

Military Teleophthalmology in Afghanistan Using Mobile Phone Application.

IMPORTANCE: The coronavirus disease 2019 pandemic has highlighted the need to expand telemedicine solutions. OBJECTIVE: To beta test a secure teleophthalmology mobile app at military treatment facilities in Afghanistan. DESIGN, SETTING, AND PARTICIPANTS: This prospective case series included 16 military treatment facilities at diverse roles of care including forward operating bases in Afghanistan and 1 location outside of Afghanistan. Thirty point-of-care medics and medical professionals were included from September to November 2019. INTERVENTIONS: Users placed teleophthalmology consults on their mobile phone using the mobile eye care app, and an expeditionary ophthalmologist stationed at a military hospital in Afghanistan responded. Users graded the mobile app using a rating scale from 1 to 5, with 1 being very dissatisfied and 5 being very satisfied. MAIN OUTCOMES AND MEASURES: Mean initial response time, agreement between the teleophthalmology diagnosis and final diagnosis, treatment and management following recommendations outlined in the Joint Trauma System clinical practice guidelines, prevention of the need for aeromedical evacuation, user satisfaction, and security and the Health Insurance Portability and Accountability Act compliance of consult. RESULTS: There were 28 consults placed over 6 weeks by 18 different users that were received by the expeditionary ophthalmologist. The mean (SD) patient age was 30.3 (9.8) years. Most patients were male (26 [93%]) and active duty US military (22 [78%]). The mean initial response time was 3 minutes 58 seconds (95% CI, 2 minutes 30 seconds to 5 minutes 26 seconds). There was agreement between the teleophthalmology diagnosis and final diagnosis in 24 consults (86%; 95% CI, 72%-100%). The treatment and management followed recommendations outlined in the Joint Trauma System Clinical Practice Guidelines for Eye Trauma: Initial Care in 28 consults (100%). Teleophthalmology consultation prevented the need for aeromedical evacuation in 4 consults (14%; 95% CI, 0.7%-28%). The patient returned to duty in 15 consults (54%; 95% CI, 34%-73%). Median overall satisfaction was 5 (minimum, 3; maximum, 5). All 28 consults (100%) were secure and compliant with the Health Insurance Portability and Accountability Act. CONCLUSIONS AND RELEVANCE: While only a limited number of consults were evaluated, this study suggests that teleophthalmology mobile phone apps may improve and extend ophthalmic care in combat zones.

Transcriptomic analysis identifies a role of PI3K-Akt signalling in the responses of skeletal muscle to acute hypoxia in vivo.

KEY POINTS: Changes in gene expression that occur within hours of exposure to hypoxia in in vivo skeletal muscles remain unexplored. Two hours of hypoxia caused significant down-regulation of extracellular matrix genes followed by a shift at 6 h to altered expression of genes associated with the nuclear lumen while respiratory and blood gases were stabilized. Enrichment analysis of mRNAs classified by stability rates suggests an attenuation of post-transcriptional regulation within hours of hypoxic exposure, where PI3K-Akt signalling was suggested to have a nodal role by pathway analysis. Experimental measurements and bioinformatic analyses suggested that the dephosphorylation of Akt after 2 h of hypoxic exposure might deactivate RNA-binding protein BRF1, hence resulting in the selective degradation of mRNAs. ABSTRACT: The effects of acute hypoxia have been widely studied, but there are few studies of transcriptional responses to hours of hypoxia in vivo, especially in hypoxia-tolerant tissues like skeletal muscles. We used RNA-seq to analyse gene expression in plantaris muscles while monitoring respiration, arterial blood gases, and blood glucose in mice exposed to 8% O2 for 2 or 6 h. Rapid decreases in blood gases and a slower reduction in blood glucose suggest stress, which was accompanied by widespread changes in gene expression. Early down-regulation of genes associated with the extracellular matrix was followed by a shift to genes associated with the nuclear lumen. Most of the early down-regulated genes had mRNA half-lives longer than 2 h, suggesting a role for post-transcriptional regulation. These transcriptional changes were enriched in signalling pathways in which the PI3K-Akt signalling pathway was identified as a hub. Our analyses indicated that gene targets of PI3K-Akt but not HIF were enriched in early transcriptional responses to hypoxia. Among the PI3K-Akt targets, 75% could be explained by a deactivation of adenylate-uridylate-rich element (ARE)-binding protein BRF1, a target of PI3K-Akt. Consistent decreases in the phosphorylation of Akt and BRF1 were experimentally confirmed following 2 h of hypoxia. These results suggest that the PI3K-Akt signalling pathway might play a role in responses induced by acute hypoxia in skeletal muscles, partially through the dephosphorylation of ARE-binding protein BRF1.

A Protocol to Collect Specific Mouse Skeletal Muscles for Metabolomics Studies.

Due to the highly sensitive nature of metabolic states, the quality of metabolomics data depends on the suitability of the experimental procedure. Metabolism could be affected by factors such as the method of euthanasia of the animals and the sample collection procedures. The effects of these factors on metabolites are tissue-specific. Thus, it is important to select proper methods to sacrifice the animal and appropriate procedures for collecting samples specific to the tissue of interest. Here, we present our protocol to collect specific mouse skeletal muscles with different fiber types for metabolomics studies. We also provide a protocol to measure lactate levels in tissue samples as a way to estimate the metabolic state in collected samples.

HIF1α Represses Cell Stress Pathways to Allow Proliferation of Hypoxic Fetal Cardiomyocytes.

Transcriptional mediators of cell stress pathways, including HIF1α, ATF4, and p53, are key to normal development and play critical roles in disease, including ischemia and cancer. Despite their importance, mechanisms by which pathways mediated by these transcription factors interact with one another are not fully understood. In addressing the controversial role of HIF1α in cardiomyocytes (CMs) during heart development, we discovered a mid-gestational requirement for HIF1α for proliferation of hypoxic CMs, involving metabolic switching and a complex interplay among HIF1α, ATF4, and p53. Loss of HIF1α resulted in activation of ATF4 and p53, the latter inhibiting CM proliferation. Bioinformatic and biochemical analyses revealed unexpected mechanisms by which HIF1α intersects with ATF4 and p53 pathways. Our results highlight previously undescribed roles of HIF1α and interactions among major cell stress pathways that could be targeted to enhance proliferation of CMs in ischemia and may have relevance to other diseases, including cancer.

MAAMD: a workflow to standardize meta-analyses and comparison of affymetrix microarray data.

BACKGROUND: Mandatory deposit of raw microarray data files for public access, prior to study publication, provides significant opportunities to conduct new bioinformatics analyses within and across multiple datasets. Analysis of raw microarray data files (e.g. Affymetrix CEL files) can be time consuming, complex, and requires fundamental computational and bioinformatics skills. The development of analytical workflows to automate these tasks simplifies the processing of, improves the efficiency of, and serves to standardize multiple and sequential analyses. Once installed, workflows facilitate the tedious steps required to run rapid intra- and inter-dataset comparisons. RESULTS: We developed a workflow to facilitate and standardize Meta-Analysis of Affymetrix Microarray Data analysis (MAAMD) in Kepler. Two freely available stand-alone software tools, R and AltAnalyze were embedded in MAAMD. The inputs of MAAMD are user-editable csv files, which contain sample information and parameters describing the locations of input files and required tools. MAAMD was tested by analyzing 4 different GEO datasets from mice and drosophila.MAAMD automates data downloading, data organization, data quality control assesment, differential gene expression analysis, clustering analysis, pathway visualization, gene-set enrichment analysis, and cross-species orthologous-gene comparisons. MAAMD was utilized to identify gene orthologues responding to hypoxia or hyperoxia in both mice and drosophila. The entire set of analyses for 4 datasets (34 total microarrays) finished in ~ one hour. CONCLUSIONS: MAAMD saves time, minimizes the required computer skills, and offers a standardized procedure for users to analyze microarray datasets and make new intra- and inter-dataset comparisons.

Using Kepler for Tool Integration in Microarray Analysis Workflows.

Increasing numbers of genomic technologies are leading to massive amounts of genomic data, all of which requires complex analysis. More and more bioinformatics analysis tools are being developed by scientist to simplify these analyses. However, different pipelines have been developed using different software environments. This makes integrations of these diverse bioinformatics tools difficult. Kepler provides an open source environment to integrate these disparate packages. Using Kepler, we integrated several external tools including Bioconductor packages, AltAnalyze, a python-based open source tool, and R-based comparison tool to build an automated workflow to meta-analyze both online and local microarray data. The automated workflow connects the integrated tools seamlessly, delivers data flow between the tools smoothly, and hence improves efficiency and accuracy of complex data analyses. Our workflow exemplifies the usage of Kepler as a scientific workflow platform for bioinformatics pipelines.

Species differences in anxiety-related responses in male prairie and meadow voles: the effects of social isolation.

Prairie (Microtus ochrogaster) and meadow voles (M. pennsylvanicus) are closely related species that differ in life strategy and social behaviors, and thus provide an excellent comparative model for the study of neuronal and hormonal mechanisms underlying behavior. In the present study using the elevated plus maze (EPM) test, we found that male prairie voles entered the open arms of the EPM more and remained there longer, and showed a higher level of overall locomotor activity than did male meadow voles. In addition, two weeks of social isolation induced an increase in open arm entries in prairie, but not meadow, voles. Prairie voles also had a higher level of circulating corticosterone compared to meadow voles, and the EPM test increased circulating corticosterone in prairie voles. Finally, social isolation coupled with the EPM test influenced Fos-immunoreactive expression in several brain areas, including the medial preoptic area, ventromedial hypothalamus, amygdala, and prefrontal cortex differently between the two species. Together, these data indicate a neural circuit involved in mediating anxiety-associated behavior in voles, and that the functioning of this circuit is influenced by social environment differently between social and non-social species.

Modulation of morphine analgesia by site-specific N-methyl-D-aspartate receptor antagonists: dependence on sex, site of antagonism, morphine dose, and time.

Pharmacological blockade of N-methyl-D-aspartate (NMDA) receptors can modulate morphine analgesia in experimental animals and humans. However, this literature is highly inconsistent, with NMDA receptor antagonists variously shown to potentiate, attenuate or produce no effect on morphine analgesic magnitude. A number of factors influencing this modulation have been proposed, but no one has examined such factors simultaneously, and all existing studies in mice were conducted exclusively in male subjects. Thus, the influence of systemic administration of site-specific NMDA receptor antagonists-including dextromethorphan, dextrorphan, MK-801, LY235959, L-701,324, and Ro 25-6981-on morphine analgesia (15-45 mg/kg; 15, 30 and 60 min post-injection) was studied in male and female mice using the 49 degrees C tail-withdrawal test. We found that oral and intraperitoneal dextromethorphan, a low-affinity non-competitive antagonist, dose-dependently potentiated low-dose morphine analgesia but attenuated high-dose morphine analgesia. Dextrorphan and MK-801 were found to potentiate low- but not high-dose morphine analgesia. The competitive glutamate-site antagonist, LY235959, and glycine-site antagonist, L-701,324, potentiated morphine analgesia at all doses. In contrast, the polyamine (NR2B) site antagonist, Ro 25-6981, attenuated morphine analgesia at all doses. Strikingly, the non-competitive antagonists produced no modulation of morphine analgesia whatsoever in female mice, whereas no sex differences were observed using competitive or NR2B antagonists. These findings indicate that NMDA modulation of morphine analgesia is critically influenced by sex, site of antagonism, morphine dose and time after injection. Our data suggest that NMDA antagonism via competitive or glycine site antagonism might result in more reliable clinical effects on morphine analgesia in both sexes.

Estrogen influences stimulated water intake by ovariectomized female rats.

To further elucidate the influence of estrogen on water consumption, we examined water intake by adult female rats stimulated by water deprivation, injection of hypertonic saline or injection of isoproterenol (ISOP), a beta-adrenergic agonist that activates the renin-angiotensin system (RAS). Rats were ovariectomized (OVX) then injected with estradiol benzoate (EB; 10 microg/0.1 ml oil) or the oil vehicle (OIL; 0.1 ml) for 2 consecutive days. Twenty-four hours after the second injection, rats were deprived of food and water. On the following day, rats were given water and intake was measured after 2 h. EB significantly decreased water intake compared with that by OIL-treated rats following water deprivation. Two additional groups of adult female rats were OVX and treated with EB or OIL. Forty-eight hours after EB or OIL treatment, rats were injected with hypertonic saline (1 ml of 2 M NaCl) or ISOP (30 microg/kg in 0.15 M saline) and water intake was measured after 2 h. EB significantly attenuated water intake following ISOP but not after hypertonic saline. Finally, we examined plasma sodium concentration (pNa) after hypertonic saline and plasma renin activity (PRA) after ISOP in EB- and OIL-treated rats and found no differences in pNa or PRA. These results suggest that the stimuli for water intake after hypertonic saline and ISOP were comparable in EB- and OIL-treated rats. Taken together, these results raise the possibility that EB attenuation of stimulated water intake is specific to water intake elicited by activation of the RAS.