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IntroductionProcedural sedation and analgesia (PSA) is accepted as a standard of care in emergency departments (ED). PSA requires careful monitoring of a patient's cardio-respiratory status, and an ability to act immediately and appropriately in the event of any untoward event. The knowledge and skills necessary for this are a natural extension of the expertise of Advanced Care Paramedics (ACP). We report a series of PSA's conducted by ACPs over a 19 month period at a busy teaching hospital. MethodsThis is a retrospective descriptive study presenting data from a registry recording details of all cases of ACP-facilitated PSA conducted in our ED between August 1, 2004 and February 28, 2006. Baseline characteristics, indication for the procedure, medications used and adverse events are reported. Results1334 ACP-facilitated PSAs were conducted during the period. According to definitions used by this study, 'desaturation'- a SaO2 of < 90% at any time during the procedure in patients with an initial SaO2 of >/- 95% occurred in only 11 (0.9%) patients, and 'hypotension' -- systolic blood pressure (SBP) < 85 mm Hg in patients with an initial SBP >/- 100 mm Hg occurred in 0.6% of patients. One significant adverse event was recorded, that of pulmonary aspiration. Medications used for PSA included fentanyl (94.1% of cases), propofol (65.5%), midazolam (36.7%) and ketamine (2.2%). ConclusionsPSA conducted in the ED by specifically trained ACPs is not associated with a significant number of adverse effects. This role should be recognized and subjected to further study. [ABSTRACT FROM AUTHOR]

OBJECTIVES: To examine the safety of emergency department (ED) procedural sedation and analgesia (PSA) and the patterns of use of pharmacologic agents at a Canadian adult teaching hospital. METHODS: Retrospective analysis of the PSA records of 979 patients, treated between Aug. 1, 2004, and July 31, 2005, with descriptive statistical analysis. This represents an inclusive consecutive case series of all PSAs performed during the study period. RESULTS: Hypotension (systolic blood pressure < or = 85 mm Hg) was documented during PSA in 13 of 979 patients (1.3%; 95% confidence interval [CI] 0.3%-2.3%), and desaturation (SaO2 < or = 90) in 14 of 979 (1.4%; Cl 0.1%-2.7%). No cases of aspiration, endotracheal intubation or death were recorded. The most common medication used was fentanyl (94.0% of cases), followed by propofol (61.2%), midazolam (42.5%) and then ketamine (2.7%). The most frequently used 2-medication combinations were propofol and fentanyl (P/F) followed by midazolam and fentanyl (M/F), used with similar frequencies 58.1% (569/979) and 41.0% (401/979) respectively. There was no significant difference in the incidence of hypotension or desaturation between the P/F and M/F treated groups. In these patients, 9.1% (90/979) of patients received more than 2 different drugs. CONCLUSIONS: Adverse events during ED PSA are rare and of doubtful clinical significance. Propofol/fentanyl and midazolam/fentanyl are used safely, and at similar frequencies for ED PSA in this tertiary hospital case series. The use of ketamine for adult PSA is unusual in our facility. [ABSTRACT FROM AUTHOR]

Unique ethical issues arise in the practice of emergency medicine, and common ethical problems are often more difficult to address in the emergency department than in other medical settings. This article is Part 2 of the Series 'Ethics in the Trenches' and it presents and analyses 2 cases - each dealing with an ethical challenge that emergency physicians are likely to encounter. The first case deals with patient refusal of care. When a patient refuses recommended care, the emergency physician must ensure the patient's decision is informed and that the patient comprehends the implications of his or her choice. The second case deals with patient involvement in criminal activities. Emergency physicians often encounter patients who have engaged in illegal activities. Al-though certain activities must be reported, physicians should be mindful of their responsibility to protect patient privacy and confidentiality. [ABSTRACT FROM AUTHOR]

Introduction: The joint Canadian Infectious Diseases Society and Canadian Thoracic Society guidelines for community-acquired pneumonia (CAP) recommend 48-72 hour telephone follow-up of patients discharged from the emergency department (ED). The guidelines provide no evidence supporting this practice, and neither the clinical utility nor the effectiveness of such recommendations has been assessed. Our objective was to assess the utility of a 48-72 hour telephone follow-up protocol for patients discharged from the ED with CAP.Methods: This was a retrospective chart audit covering a 2-year period (Jan. 3, 1999 to Jan. 3, 2001) after the introduction of a clinical practice guideline (CPG) that included routine 48-72 hour telephone follow-up of patients discharged from the ED with CAP. Eligible patients were identified in the ED database, rates of referral for telephone follow-up were recorded, and 30-day outcomes (death and readmission) for patients referred versus not referred were compared.Results: During the study period, 867 patients were identified as being eligible for the study. The mean age was 55.7 years (range 16-98 yr), and mean pneumonia severity index (PSI) was 68.9 (range 6-187). Despite the CPG, only 148 patients (17.1%) were referred for telephone follow-up. Age, demographics, comorbidity, clinical status and pneumonia severity were similar for referred and non-referred patients. Thirty-day death (2.5%) and readmission rates (3%) were strongly related to PSI score, but did not differ significantly in the 2 comparison groups.Conclusion: In this setting, physicians were poorly compliant with a routine telephone follow-up protocol. The likelihood of referral for follow-up did not correlate with pneumonia severity, and follow-up referral did not appear to affect patient outcome. These findings do not support recommendations for routine early follow-up mechanisms beyond those already existing in the community. [ABSTRACT FROM AUTHOR]

Study Objective: To assess the clinical usefulness of blood cultures (BCs) in the management of patients hospitalized with community-acquired pneumonia (CAP).Design: A prospective, observational study to investigate the contribution of BCs to the management and outcomes of adult patients presenting with CAP.Setting: Nineteen Canadian hospitals.Patients: Adults admitted to the hospital with CAP between January 1, 1998, and July 31, 1998.Interventions: The courses of therapy in patients for whom BC results yielded organisms considered to be clinically significant were analyzed to determine whether the BCs had contributed to management or outcome.Measurements and Results: Forty-three of 760 patients had significantly positive BC results. Patients with CAP who had BCs performed had a 1.97% chance (15 of 760 patients) of having a change of therapy directed by BC results. Patients in whom BCs yielded positive results had a 34.8% chance (15 of 43 patients) of having a change in therapy determined by BC results, and had a 58.1% chance (25 of 43 patients) of having a course of therapy contraindicated by BC results. Severity of illness, as measured by the pneumonia severity index, correlated poorly with the yield of BCs. BC results were positive in 8.0% of patients in risk classes I and II, 6.2% of patients in risk class III, 4.6% of patients in risk class IV, and 5.2% of patients in risk class V.Conclusion: BCs have limited usefulness in the routine management of patients admitted to the hospital with uncomplicated CAP. [ABSTRACT FROM AUTHOR]

The issue of error in medicine has attracted worldwide attention in the past few years. Initiatives to define the nature and full extent of the problem have failed, thus far, to address the issues particular to rural medicine. This article provides an overview of what is known about medical error in this setting and suggests strategies by which the rural medical community can approach this important subject. [ABSTRACT FROM AUTHOR]

OBJECTIVES To assess whether there is: a) a clinical difference between patients with cellulitis treated according to the recommendations of a clinical cellulitis guideline, and those treated otherwise and b) a difference in the cost of antibiotic treatment of the two groups. METHODS Emergency Department (ED) patients diagnosed with cellulitis at a community hospital were graded using a 4-point scale, and physicians were encouraged to treat based on an established practice guideline. Patients were contacted 5 days after their ED visit, and again at 10 days if they had not improved by 5 days. Physician 'compliance' was defined as having followed three or more of the five elements of the guideline. RESULTS Of the 272 patients, 147 (54.1%) were classified as Grade I, 53 (19.5%) Grade II, 33(12.1%) Grade III, and 6 (2.2%) Grade IV. In 12.1% the grade was not assigned. 43.5% were treated in compliance with the guidelines, of which 83.3% reported improvement at 5 days, compared to 87.7% of those treated otherwise. At 10 days, 98.8% of the patients treated in compliance with the protocol had improved compared to 94.7% of those treated otherwise. Average antibiotic cost/patient was: Grade 1: $8.48, Grade II: $16.65, Grade II: $96.53 in the 'compliance' group, and $35.68, $51.28, and $150.18 respectively in the 'non-compliance' group. CONCLUSIONS Patients treated in accordance with the cellulitis guideline had similar outcomes to those treated otherwise, at significantly lower cost. Efforts to encourage compliance with the guideline are indicated. [ABSTRACT FROM AUTHOR]

Hypertrophic and dilated cardiomyopathies (HCM and DCM, respectively) are inherited disorders that may be caused by mutations to the same sarcomeric protein but have completely different clinical phenotypes. The precise mechanisms by which point mutations within the same gene bring about phenotypic diversity remain unclear. Our objective has been to develop a mechanistic explanation of diverging phenotypes in two TPM1 mutations, E62Q (HCM) and E54K (DCM). Drawing on data from the literature and experiments with stem cell-derived cardiomyocytes expressing the TPM1 mutations of interest, we constructed computational simulations that provide plausible explanations of the distinct muscle contractility caused by each variant. In E62Q, increased calcium sensitivity and hypercontractility was explained most accurately by a reduction in effective molecular stiffness of tropomyosin and alterations in its interactions with the actin thin filament that favor the 'closed' regulatory state. By contrast, the E54K mutation appeared to act via long-range allosteric interactions to increase the association rate of the C-terminal troponin I mobile domain to tropomyosin/actin. These mutation-linked molecular events produced diverging alterations in gene expression that can be observed in human engineered heart tissues. Modulators of myosin activity confirmed our proposed mechanisms by rescuing normal contractile behavior in accordance with predictions.

Eukaryotic initiation factor 2B (eIF2B) controls translation initiation by recycling inactive eIF2-GDP to active eIF2-GTP. Under cellular stress, the integrated stress response (ISR) is activated inhibiting eIF2B activity resulting in the translation attenuation and reprogramming of gene expression to overcome the stress. The ISR can dictate cell fate wherein chronic activation has pathological outcomes. Vanishing white matter disease (VWMD) is a chronic ISR-related disorder with mutations in eIF2B targeting astrocyte and oligodendrocyte cells. Regulation of eIF2B localization (eIF2B bodies) has been implicated in the ISR. We present evidence that neuronal and glial cell types possess distinct patterns of eIF2B bodies which change in a manner correlating to acute and chronic ISR activation. We also demonstrate that while neural and glial cell types respond similarly to the acute induction of the ISR a chronic ISR exerts cell-type specific differences. These findings provide key insights into neural cell responses and adaptation to cellular stress., Competing Interests: For the purpose of open access, the author has applied a Creative Commons Attribution (CC BY) license to any Author Accepted article version of this article arising from this submission. The authors have no relevant financial or non-financial interests to disclose. The author Dr Filipe Hanson completed this work as part of his PhD studies at Sheffield Hallam University. He now works as a Research Scientist for UCB Pharma, Slough, UK. The author Dr Madalena deOliveira contributed to this work as part of her PhD studies at Sheffield Hallam University. She now works as a Postdoctoral Fellow for MSD, UK., (© 2024 The Author(s).)

Dilated cardiomyopathy (DCM) is an inherited disorder often leading to severe heart failure. Linkage studies in affected families have revealed hundreds of different mutations that can cause DCM, with most occurring in genes associated with the cardiac sarcomere. We have developed an investigational pipeline for discovering mechanistic genotype-phenotype relationships in DCM and here apply it to the DCM-linked tropomyosin mutation TPM1 M8R. Atomistic simulations predict that M8R increases flexibility of the tropomyosin chain and enhances affinity for the blocked or inactive state of tropomyosin on actin. Applying these molecular effects to a Markov model of the cardiac thin filament reproduced the shifts in Ca 2+ sensitivity, maximum force, and a qualitative drop in cooperativity that were observed in an in vitro system containing TPM1 M8R. The model was then used to simulate the impact of M8R expression on twitch contractions of intact cardiac muscle, predicting that M8R would reduce peak force and duration of contraction in a dose-dependent manner. To evaluate this prediction, TPM1 M8R was expressed via adenovirus in human engineered heart tissues and isometric twitch force was observed. The mutant tissues manifested depressed contractility and twitch duration that agreed in detail with model predictions. Additional exploratory simulations suggest that M8R-mediated alterations in tropomyosin-actin interactions contribute more potently than tropomyosin chain stiffness to cardiac twitch dysfunction, and presumably to the ultimate manifestation of DCM. This study is an example of the growing potential for successful in silico prediction of mutation pathogenicity for inherited cardiac muscle disorders., Competing Interests: Yale University has patented and licensed technology used in this research to a commercial entity (Propria LLC). SC is founder and holds an equity stake in Propria LLC. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Creso, Gokhan, Rynkiewicz, Lehman, Moore and Campbell.)

Originally developed more than 20 years ago, engineered heart tissue (EHT) has become an important tool in cardiovascular research for applications such as disease modeling and drug screening. Innovations in biomaterials, stem cell biology, and bioengineering, among other fields, have enabled EHT technologies to recapitulate many aspects of cardiac physiology and pathophysiology. While initial EHT designs were inspired by the isolated-trabecula culture system, current designs encompass a variety of formats, each of which have unique strengths and limitations. In this review, we describe the most common EHT formats, and then systematically evaluate each aspect of their design, emphasizing the rational selection of components for each application., Competing Interests: S.G.C. has equity ownership in Propria, LLC, which develops engineered heart tissue technology., (© 2024 Author(s).)

Understanding how cognitive biases, mental models, and mindsets impact leadership in health systems is essential. This article supports the notion of cognitive biases as flawed thinking or cognitive traps which negatively influence leadership. Mental models that do not fit with current evidence limit our ability to comprehend and respond to system issues. Resulting mindsets affect cognition, behaviour, and decision-making. Metacognition is critical. The wicked problems in today's complex health system require leaders and everyone involved to elevate their personal, organizational, and disciplinary perspectives to a systems level. Three examples of mental models/mindsets are reviewed. They do not change simply because we wish or will them to. The first step is being aware of what they are and how they impact our thinking and decision-making. Some tips for managing these traps are offered as examples of how to challenge our leadership approach in the health system., Competing Interests: Declaration of conflicting interestsThe author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Titin (TTN) is one of the largest and most complex proteins expressed in humans, and truncation variants are the most prevalent genetic lesion identified in individuals with dilated cardiomyopathy (DCM) or other disorders of impaired cardiac contractility. Two reports in this issue of the JCI shed light on a potential mechanism involving truncated TTN sarcomere integration and the potential for disruption of sarcomere structural integrity. Kellermayer, Tordai, and colleagues confirmed the presence of truncated TTN protein in human DCM samples. McAfee and authors developed a patient-specific TTN antibody to study truncated TTN subcellular localization and to explore its functional consequences. A "poison peptide" mechanism emerges that inspires alternative therapeutic approaches while opening new lines for inquiry, such as the role of haploinsufficiency of full-length TTN protein, mechanisms explaining sarcomere dysfunction, and explanations for variable penetrance.

Introduction Certain patient groups perceive specific barriers to accessing primary care, resulting in increased emergency department (ED) use for non-emergency conditions. There is evidence coming from other countries that homeless people are treated differently in accessing emergency services. Examination of ED wait time by demographic characteristics provides pertinent information to identify subgroups that are more subject to the consequences or causes of access block and delayed treatment.  Methods We analyzed five years of Emergency Department Information System (EDIS) visit records of the largest tertiary care center in Atlantic Canada. The wait time from triage to seeing a physician was the outcome, housing status was the main exposure, and age and gender were the predictors. Quantile regressions were carried out to identify the influence of homeless visits in meeting the Canadian Association of Emergency Physicians (CAEP) wait time benchmarks for each Canadian Triage and Acuity Scale (CTAS) level. The classification and regression tree method was used to quantify and classify the demographic subgroups contributing to wait time disparities across CTAS levels. Results Homeless visit median wait times that exceeded the three-hour CAEP benchmark were significantly longer for urgent (by 40 minutes, CI: 25-55), semi-urgent (by 31 minutes, CI: 17-45), and non-urgent (by 57 minutes, CI: 25-89) than acuity level-matched domiciled visit wait times. At the 50th percentile, one-hour benchmark homeless triaged as semi-urgent waited (median=20 minutes, CI: 12-28) longer, and no other triage-level differences were found at this benchmark. Homeless emergent-level visits that exceeded the three-hour benchmark were 28 minutes, on average, shorter than domiciled patients of the same acuity level. Homeless females above 40 stayed the longest for non-urgent care (mean=173 minutes), 82 minutes longer (p=0.0001) than age-gender-acuity level-matched domiciled patients. Homelessness was the most prominent ED wait time classifier for non-urgent, ED visits. Overall, homeless patients triaged as CTAS-5 waited 30 minutes longer (p=0.0001) than domiciled patients triaged as CTAS-5. Homeless male 16-20-year-olds waited the shortest time of 72 minutes.  Conclusion Homelessness-related wait time disparities exist in the low acuity non-urgent-level ED visits more than in the other levels, supporting the theory that lack of primary care access is a driver of ED use in this group. Our acuity level analysis supports that homeless people of a certain age (older) and gender groups (female) wait longer than their age-gender-matched domiciled patients to be seen by a physician in low acuity level presentations. Given the pattern of the homeless being seen earlier or statistically similar in emergent-level visits compared to matched domiciled patients and that 16-20-year-old homeless males were seen on average within 72 minutes (the shortest mean wait time reported for the triage level CTAS-5), we decline the notion of discrimination at the study site ED. If homeless patients' non-urgent needs were met elsewhere, pressure on the ED to meet benchmarks might be reduced., Competing Interests: The authors have declared that no competing interests exist., (Copyright © 2023, Weerasinghe et al.)

We seek to elucidate the precise nature of mechanical loading that precipitates conduction deficits in a concealed-phase model of arrhythmogenic cardiomyopathy (ACM). ACM is a progressive disorder often resulting from mutations in desmosomal proteins. Exercise has been shown to worsen disease progression and unmask arrhythmia vulnerability, yet the underlying pathomechanisms may depend on the type and intensity of exercise. Because exercise causes myriad changes to multiple inter-dependent hemodynamic parameters, it is difficult to isolate its effects to specific changes in mechanical load. Here, we use engineered heart tissues (EHTs) with iPSC-derived cardiomyocytes expressing R451G desmoplakin, an ACM-linked mutation, which results in a functionally null model of desmoplakin (DSP). We also use a novel bioreactor to independently perturb tissue strain at different time points during the cardiac cycle. We culture EHTs under three strain regimes: normal physiological shortening; increased diastolic stretch, simulating high preload; and isometric culture, simulating high afterload. DSP R451G EHTs that have been cultured isometrically undergo adaptation, with no change in action potential parameters, conduction velocity, or contractile function, a phenotype confirmed by global proteomic analysis. However, when DSP R451G EHTs are subjected to increased diastolic stretch, they exhibit concomitant reductions in conduction velocity and the expression of connexin-43. These effects are rescued by inhibition of both lysosome activity and ERK signaling. Our results indicate that the response of DSP R451G EHTs to mechanical stimuli depends on the strain and the timing of the applied stimulus, with increased diastolic stretch unmasking conduction deficits in a concealed-phase model of ACM.

Decellularized porcine myocardium is commonly used as scaffolding for engineered heart tissues (EHTs). However, structural and mechanical heterogeneity in the myocardium complicate production of mechanically consistent tissues. In this study, we evaluate the porcine psoas major muscle (tenderloin) as an alternative scaffold material. Head-to-head comparison of decellularized tenderloin and ventricular scaffolds showed only minor differences in mean biomechanical characteristics, but tenderloin scaffolds were less variable and less dependent on the region of origin than ventricular samples. The active contractile behavior of EHTs made by seeding tenderloin versus ventricular scaffolds with human-induced pluripotent stem cell-derived cardiomyocytes was also comparable, with only minor differences observed. Collectively, the data reveal that the behavior of EHTs produced from decellularized porcine psoas muscle is almost identical to those made from porcine left ventricular myocardium, with the advantages of being more homogeneous, biomechanically consistent, and readily obtainable.

Background and aims Choosing Wisely Nova Scotia (CWNS), an affiliate of Choosing Wisely Canada (CWC), aims to address unnecessary care and tests through literature-informed lists developed by various disciplines. CWC has identified unnecessary head CTs among the top five tests, procedures, and treatments to question within the emergency department setting. The Canadian CT-scan Head Rule (CCHR) has been found to be the most effective clinical decision rule in adults with minor head injuries. This study aimed to better understand the current status of CCHR use in Nova Scotia, we conducted a retrospective audit of patient charts at the Charles V. Keating Emergency and Trauma Center in Halifax, Nova Scotia. Materials and methods Our mixed methods design included a narrative literature review, a retrospective chart audit, and a qualitative audit-feedback component with physicians who work in the emergency department (ED). The chart audit applied the guidelines for adherence to the CCHR and reported on the level of compliance within the ED. Results Analysis of qualitative data is included here, in parallel with in-depth analysis to contextualize findings from the chart audit. A total of 302 charts of patients presenting to the surveyed site were retrospectively reviewed for this study. Of the 37 cases where the CT head was indicated as per the CCHR, a CT was ordered 32 (86.5%) times. Of the 176 cases where a CT head was not indicated as per the CCHR, a CT was not ordered 155 (88.1%) times. Therefore, the CCHR was followed in 187 (87.8%) of the total 213 cases where the CCHR should be applied. Conclusions Our review revealed that the CCHR was adhered in 87.8% of cases at the surveyed ED. Identifying contextual factors that facilitate or hinder the application of CCHR in practice is critical to achieving the goal of reducing unnecessary CTs. This work will be presented to the physician group to engage and understand factors that are enablers in the process of ED minor head injury care., Competing Interests: The authors have declared that no competing interests exist., (Copyright © 2023, Sampalli et al.)

Engineered whole lungs may one day expand therapeutic options for patients with end-stage lung disease. However, the feasibility of ex vivo lung regeneration remains limited by the inability to recapitulate mature, functional alveolar epithelium. Here, we modulate multimodal components of the alveolar epithelial type 2 cell (AEC2) niche in decellularized lung scaffolds in order to guide AEC2 behavior for epithelial regeneration. First, endothelial cells coordinate with fibroblasts, in the presence of soluble growth and maturation factors, to promote alveolar scaffold population with surfactant-secreting AEC2s. Subsequent withdrawal of Wnt and FGF agonism synergizes with tidal-magnitude mechanical strain to induce the differentiation of AEC2s to squamous type 1 AECs (AEC1s) in cultured alveoli, in situ. These results outline a rational strategy to engineer an epithelium of AEC2s and AEC1s contained within epithelial-mesenchymal-endothelial alveolar-like units, and highlight the critical interplay amongst cellular, biochemical, and mechanical niche cues within the reconstituting alveolus., (© 2023. The Author(s).)

Chronic stimulation of cardiac α 1A -adrenergic receptors (α 1A -ARs) improves symptoms in multiple preclinical models of heart failure. However, the translational significance remains unclear. Human engineered heart tissues (EHTs) provide a means of quantifying the effects of chronic α 1A -AR stimulation on human cardiomyocyte physiology. EHTs were created from thin slices of decellularized pig myocardium seeded with human induced pluripotent stem cell (iPSC)-derived cardiomyocytes and fibroblasts. With a paired experimental design, EHTs were cultured for 3 wk, mechanically tested, cultured again for 2 wk with α 1A -AR agonist A61603 (10 nM) or vehicle control, and retested after drug washout for 24 h. Separate control experiments determined the effects of EHT age (3-5 wk) or repeat mechanical testing. We found that chronic A61603 treatment caused a 25% increase of length-dependent activation (LDA) of contraction compared with vehicle treatment ( n = 7/group, P = 0.035). EHT force was not increased after chronic A61603 treatment. However, after vehicle treatment, EHT force was increased by 35% relative to baseline testing ( n = 7/group, P = 0.022), suggesting EHT maturation. Control experiments suggested that increased EHT force resulted from repeat mechanical testing, not from EHT aging. RNA-seq analysis confirmed that the α 1A -AR is expressed in human EHTs and found chronic A61603 treatment affected gene expression in biological pathways known to be activated by α 1A -ARs, including the MAP kinase signaling pathway. In conclusion, increased LDA in human EHT after chronic A61603 treatment raises the possibility that chronic stimulation of the α 1A -AR might be beneficial for increasing LDA in human myocardium and might be beneficial for treating human heart failure by restoring LDA. NEW & NOTEWORTHY Chronic stimulation of α 1A -adrenergic receptors (α 1A -ARs) is known to mediate therapeutic effects in animal heart failure models. To investigate the effects of chronic α 1A -AR stimulation in human cardiomyocytes, we tested engineered heart tissue (EHT) created with iPSC-derived cardiomyocytes. RNA-seq analysis confirmed human EHT expressed α 1A -ARs. Chronic (2 wk) α 1A -AR stimulation with A61603 (10 nM) increased length-dependent activation (LDA) of contraction. Chronic α 1A -AR stimulation might be beneficial for treating human heart failure by restoring LDA.

Porphyromonas gingivalis contributes to the chronic oral disease periodontitis, triggering the activation of host inflammatory responses, inducing cellular stresses such as oxidation. During stress, host cells can activate the Integrated Stress Response (ISR), a pathway which determines cellular fate, by either downregulating protein synthesis and initiating a stress-response gene expression program, or by initiating programmed cell death. Recent studies have implicated the ISR within both host antimicrobial defenses and the pathomechanism of certain microbes. In this study, using a combination of immunofluorescence confocal microscopy and immunoblotting, the molecular mechanisms by which P. gingivalis infection alters translation attenuation during oxidative stress-induced activation of the ISR in oral epithelial cells were investigated. P. gingivalis infection alone did not result in ISR activation. In contrast, infection coupled with stress caused differential stress granule formation and composition. Infection heightened stress-induced translational repression independently of core ISR mediators. Heightened translational repression during stress was observed with both P. gingivalis -conditioned media and outer membrane vesicles, implicating a secretory factor in this exacerbated translational repression. The effects of gingipain inhibitors and gingipain-deficient P. gingivalis mutants confirmed these pathogen-specific proteases as the effector of exacerbated translational repression. Gingipains are known to degrade the mammalian target of rapamycin (mTOR) and the findings of this study implicate the gingipain-mTOR axis as the effector of host translational dysregulation during stress.

The historical tendency to view medicine as both an art and a science may have contributed to a disinclination among clinicians towards cognitive science. In particular, this has had an impact on the approach towards the diagnostic process which is a barometer of clinical decision-making behaviour and is increasingly seen as a yardstick of clinician calibration and performance. The process itself is more complicated and complex than was previously imagined, with multiple variables that are difficult to predict, are interactive, and show nonlinearity. They appear to characterise a complex adaptive system. Many aspects of the diagnostic process, including the psychophysics of signal detection and discrimination, ergonomics, probability theory, decision analysis, factor analysis, causal analysis and more recent developments in judgement and decision-making (JDM), especially including the domain of heuristics and cognitive and affective biases, appear fundamental to a good understanding of it. A preliminary analysis of factors such as manifestness of illness and others that may impede clinicians' awareness and understanding of these issues is proposed here. It seems essential that medical trainees be explicitly and systematically exposed to specific areas of cognitive science during the undergraduate curriculum, and learn to incorporate them into clinical reasoning and decision-making. Importantly, this understanding is needed for the development of cognitive bias mitigation and improved calibration of JDM in clinical practice., (© 2023. The Author(s).)

Hypertrophic cardiomyopathy (HCM) is an inherited disorder often caused by mutations to sarcomeric genes. Many different HCM-associated TPM1 mutations have been identified but they vary in their degrees of severity, prevalence, and rate of disease progression. The pathogenicity of many TPM1 variants detected in the clinical population remains unknown. Our objective was to employ a computational modeling pipeline to assess pathogenicity of one such variant of unknown significance, TPM1 S215L, and validate predictions using experimental methods. Molecular dynamic simulations of tropomyosin on actin suggest that the S215L significantly destabilizes the blocked regulatory state while increasing flexibility of the tropomyosin chain. These changes were quantitatively represented in a Markov model of thin-filament activation to infer the impacts of S215L on myofilament function. Simulations of in vitro motility and isometric twitch force predicted that the mutation would increase Ca 2+ sensitivity and twitch force while slowing twitch relaxation. In vitro motility experiments with thin filaments containing TPM1 S215L revealed higher Ca 2+ sensitivity compared with wild type. Three-dimensional genetically engineered heart tissues expressing TPM1 S215L exhibited hypercontractility, upregulation of hypertrophic gene markers, and diastolic dysfunction. These data form a mechanistic description of TPM1 S215L pathogenicity that starts with disruption of the mechanical and regulatory properties of tropomyosin, leading thereafter to hypercontractility and finally induction of a hypertrophic phenotype. These simulations and experiments support the classification of S215L as a pathogenic mutation and support the hypothesis that an inability to adequately inhibit actomyosin interactions is the mechanism whereby thin-filament mutations cause HCM., (© The Author(s) 2023. Published by Oxford University Press on behalf of National Academy of Sciences.)

Familial cardiomyopathy is a precursor of heart failure and sudden cardiac death. Over the past several decades, researchers have discovered numerous gene mutations primarily in sarcomeric and cytoskeletal proteins causing two different disease phenotypes: hypertrophic (HCM) and dilated (DCM) cardiomyopathies. However, molecular mechanisms linking genotype to phenotype remain unclear. Here, we employ a systems approach by integrating experimental findings from preclinical studies (e.g., murine data) into a cohesive signaling network to scrutinize genotype to phenotype mechanisms. We developed an HCM/DCM signaling network model utilizing a logic-based differential equations approach and evaluated model performance in predicting experimental data from four contexts (HCM, DCM, pressure overload, and volume overload). The model has an overall prediction accuracy of 83.8%, with higher accuracy in the HCM context (90%) than DCM (75%). Global sensitivity analysis identifies key signaling reactions, with calcium-mediated myofilament force development and calcium-calmodulin kinase signaling ranking the highest. A structural revision analysis indicates potential missing interactions that primarily control calcium regulatory proteins, increasing model prediction accuracy. Combination pharmacotherapy analysis suggests that downregulation of signaling components such as calcium, titin and its associated proteins, growth factor receptors, ERK1/2, and PI3K-AKT could inhibit myocyte growth in HCM. In experiments with patient-specific iPSC-derived cardiomyocytes (MLP-W4R;MYH7-R723C iPSC-CMs), combined inhibition of ERK1/2 and PI3K-AKT rescued the HCM phenotype, as predicted by the model. In DCM, PI3K-AKT-NFAT downregulation combined with upregulation of Ras/ERK1/2 or titin or Gq protein could ameliorate cardiomyocyte morphology. The model results suggest that HCM mutations that increase active force through elevated calcium sensitivity could increase ERK activity and decrease eccentricity through parallel growth factors, Gq-mediated, and titin pathways. Moreover, the model simulated the influence of existing medications on cardiac growth in HCM and DCM contexts. This HCM/DCM signaling model demonstrates utility in investigating genotype to phenotype mechanisms in familial cardiomyopathy., Competing Interests: Declaration of Competing Interest A.D.M. and J.H.O. are co-founders of and have equity interests in Insilicomed Inc. and serve as scientific advisors. A.D.M is also a co-founder of and scientific advisor to Vektor Medical, Inc. Some of their research grants have been identified for conflict of interest management based on the overall scope of the project and its potential benefit to these companies. These authors are required to disclose this relationship in publications acknowledging the grant support; however, the research subject and findings reported in this study did not involve the companies in any way and had no relationship with the business activities or scientific interests of either company. The terms of this arrangement have been reviewed and approved by the University of California San Diego in accordance with its conflict of interest policies., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Parkinson's disease (PD) is a common neurodegenerative condition affecting a significant number of individuals globally, resulting in the presentation of debilitating motor and non-motor symptoms, including bradykinesia, resting tremor, as well as mood and sleep disorders. The pathology of PD has been observed to spread through the central nervous system resulting in progressive brain degeneration and a poor prognosis. Aggregated forms of the protein α-synuclein, particularly intermediary aggregates, referred to as oligomers, or preformed fibrils, have been implicated as the causative agent in the degeneration of neuronal processes, including the dysfunction of axonal transport, mitochondrial activity, and ultimately cellular death. Extracellular vesicles (EVs) have been strongly implicated in the propagation of PD pathology. Current observations suggest that aggregated α-synuclein is transported between neurons via small EVs in a series of exocytosis and endocytosis cellular processes leading to the observed spread of neurotoxicity and cellular death. Despite some understanding of the role of EVs in neurodegeneration, the exact mechanism by which these lipidic particles participate in the progression of Parkinson's pathology is not entirely understood. Here we review the current understanding of the role of EVs in the propagation of PD and explore their potential as a therapeutic target., (© 2022 The Author(s).)

Arrhythmogenic cardiomyopathy (ACM) is an inherited disorder characterized by fibro-fatty infiltration with an increased propensity for ventricular arrhythmias and sudden death. Genetic variants in desmosomal genes are associated with ACM. Incomplete penetrance is a common feature in ACM families, complicating the understanding of how external stressors contribute towards disease development. To analyze the dual role of genetics and external stressors on ACM progression, we developed one of the first mouse models of ACM that recapitulates a human variant by introducing the murine equivalent of the human R451G variant into endogenous desmoplakin ( Dsp R451G/+ ) . Mice homozygous for this variant displayed embryonic lethality. While Dsp R451G/+ mice were viable with reduced expression of DSP, no presentable arrhythmogenic or structural phenotypes were identified at baseline. However, increased afterload resulted in reduced cardiac performance, increased chamber dilation, and accelerated progression to heart failure. In addition, following catecholaminergic challenge, Dsp R451G/+ mice displayed frequent and prolonged arrhythmic events. Finally, aberrant localization of connexin-43 was noted in the Dsp R451G/+ mice at baseline, becoming more apparent following cardiac stress via pressure overload. In summary, cardiovascular stress is a key trigger for unmasking both electrical and structural phenotypes in one of the first humanized ACM mouse models.

Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have wide potential application in basic research, drug discovery, and regenerative medicine, but functional maturation remains challenging. Here, we present a method whereby maturation of hiPSC-CMs can be accelerated by simultaneous application of physiological Ca 2+ and frequency-ramped electrical pacing in culture. This combination produces positive force-frequency behavior, physiological twitch kinetics, robust β-adrenergic response, improved Ca 2+ handling, and cardiac troponin I expression within 25 days. This study provides insights into the role of Ca 2+ in hiPSC-CM maturation and offers a scalable platform for translational and clinical research., Competing Interests: Conflicts of interest S.G.C. has equity ownership in Propria LLC, which has licensed EHT technology used in the research reported in this publication. This arrangement has been reviewed and approved by the Yale University Conflict of Interest Office. The authors declare no additional competing financial interests., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

Eukaryotic initiation factor 2B, eIF2B is a guanine nucleotide exchange, factor with a central role in coordinating the initiation of translation. During stress and disease, the activity of eIF2B is inhibited via the phosphorylation of its substrate eIF2 (p-eIF2α). A number of different kinases respond to various stresses leading to the phosphorylation of the alpha subunit of eIF2, and collectively this regulation is known as the integrated stress response, ISR. This targeting of eIF2B allows the cell to regulate protein synthesis and reprogramme gene expression to restore homeostasis. Advances within structural biology have furthered our understanding of how eIF2B interacts with eIF2 in both the productive GEF active form and the non-productive eIF2α phosphorylated form. Here, current knowledge of the role of eIF2B in the ISR is discussed within the context of normal and disease states focusing particularly on diseases such as vanishing white matter disease (VWMD) and permanent neonatal diabetes mellitus (PNDM), which are directly linked to mutations in eIF2B. The role of eIF2B in synaptic plasticity and memory formation is also discussed. In addition, the cellular localisation of eIF2B is reviewed and considered along with the role of additional in vivo eIF2B binding factors and protein modifications that may play a role in modulating eIF2B activity during health and disease., (© 2022 The Author(s).)

Background: Familial hypertrophic cardiomyopathy (HCM) is the most common inherited cardiac disease and is typically caused by mutations in genes encoding sarcomeric proteins that regulate cardiac contractility. HCM manifestations include left ventricular hypertrophy and heart failure, arrythmias, and sudden cardiac death. How dysregulated sarcomeric force production is sensed and leads to pathological remodeling remains poorly understood in HCM, thereby inhibiting the efficient development of new therapeutics., Methods: Our discovery was based on insights from a severe phenotype of an individual with HCM and a second genetic alteration in a sarcomeric mechanosensing protein. We derived cardiomyocytes from patient-specific induced pluripotent stem cells and developed robust engineered heart tissues by seeding induced pluripotent stem cell-derived cardiomyocytes into a laser-cut scaffold possessing native cardiac fiber alignment to study human cardiac mechanobiology at both the cellular and tissue levels. Coupled with computational modeling for muscle contraction and rescue of disease phenotype by gene editing and pharmacological interventions, we have identified a new mechanotransduction pathway in HCM, shown to be essential in modulating the phenotypic expression of HCM in 5 families bearing distinct sarcomeric mutations., Results: Enhanced actomyosin crossbridge formation caused by sarcomeric mutations in cardiac myosin heavy chain ( MYH7 ) led to increased force generation, which, when coupled with slower twitch relaxation, destabilized the MLP (muscle LIM protein) stretch-sensing complex at the Z-disc. Subsequent reduction in the sarcomeric muscle LIM protein level caused disinhibition of calcineurin-nuclear factor of activated T-cells signaling, which promoted cardiac hypertrophy. We demonstrate that the common muscle LIM protein-W4R variant is an important modifier, exacerbating the phenotypic expression of HCM, but alone may not be a disease-causing mutation. By mitigating enhanced actomyosin crossbridge formation through either genetic or pharmacological means, we alleviated stress at the Z-disc, preventing the development of hypertrophy associated with sarcomeric mutations., Conclusions: Our studies have uncovered a novel biomechanical mechanism through which dysregulated sarcomeric force production is sensed and leads to pathological signaling, remodeling, and hypertrophic responses. Together, these establish the foundation for developing innovative mechanism-based treatments for HCM that stabilize the Z-disc MLP-mechanosensory complex.

Background: Altered kinase localization is gaining appreciation as a mechanism of cardiovascular disease. Previous work suggests GSK-3β (glycogen synthase kinase 3β) localizes to and regulates contractile function of the myofilament. We aimed to discover GSK-3β's in vivo role in regulating myofilament function, the mechanisms involved, and the translational relevance., Methods: Inducible cardiomyocyte-specific GSK-3β knockout mice and left ventricular myocardium from nonfailing and failing human hearts were studied., Results: Skinned cardiomyocytes from knockout mice failed to exhibit calcium sensitization with stretch indicating a loss of length-dependent activation (LDA), the mechanism underlying the Frank-Starling Law. Titin acts as a length sensor for LDA, and knockout mice had decreased titin stiffness compared with control mice, explaining the lack of LDA. Knockout mice exhibited no changes in titin isoforms, titin phosphorylation, or other thin filament phosphorylation sites known to affect passive tension or LDA. Mass spectrometry identified several z-disc proteins as myofilament phospho-substrates of GSK-3β. Agreeing with the localization of its targets, GSK-3β that is phosphorylated at Y216 binds to the z-disc. We showed pY216 was necessary and sufficient for z-disc binding using adenoviruses for wild-type, Y216F, and Y216E GSK-3β in neonatal rat ventricular cardiomyocytes. One of GSK-3β's z-disc targets, abLIM-1 (actin-binding LIM protein 1), binds to the z-disc domains of titin that are important for maintaining passive tension. Genetic knockdown of abLIM-1 via siRNA in human engineered heart tissues resulted in enhancement of LDA, indicating abLIM-1 may act as a negative regulator that is modulated by GSK-3β. Last, GSK-3β myofilament localization was reduced in left ventricular myocardium from failing human hearts, which correlated with depressed LDA., Conclusions: We identified a novel mechanism by which GSK-3β localizes to the myofilament to modulate LDA. Importantly, z-disc GSK-3β levels were reduced in patients with heart failure, indicating z-disc localized GSK-3β is a possible therapeutic target to restore the Frank-Starling mechanism in patients with heart failure.

Myofilaments and their associated proteins, which together constitute the sarcomeres, provide the molecular-level basis for contractile function in all muscle types. In intact muscle, sarcomere-level contraction is strongly coupled to other cellular subsystems, in particular the sarcolemmal membrane. Skinned muscle preparations (where the sarcolemma has been removed or permeabilized) are an experimental system designed to probe contractile mechanisms independently of the sarcolemma. Over the last few decades, experiments performed using permeabilized preparations have been invaluable for clarifying the understanding of contractile mechanisms in both skeletal and cardiac muscle. Today, the technique is increasingly harnessed for preclinical and/or pharmacological studies that seek to understand how interventions will impact intact muscle contraction. In this context, intrinsic functional and structural differences between skinned and intact muscle pose a major interpretational challenge. This review first surveys measurements that highlight these differences in terms of the sarcomere structure, passive and active tension generation, and calcium dependence. We then highlight the main practical challenges and caveats faced by experimentalists seeking to emulate the physiological conditions of intact muscle. Gaining an awareness of these complexities is essential for putting experiments in due perspective., (© 2022 Lewalle et al.)

There is a need for improved 3-dimensional (3D) lung models that recapitulate the architectural and cellular complexity of the native lung alveolus ex vivo. Recently developed organoid models have facilitated the expansion and study of lung epithelial progenitors in vitro, but these platforms typically rely on mouse tumor-derived matrix and/or serum, and incorporate just one or two cellular lineages. Here, we describe a protocol for generating engineered lung tissues (ELTs) based on the multi-lineage recellularization of decellularized precision-cut lung slices (PCLS). ELTs contain alveolar-like structures comprising alveolar epithelium, mesenchyme, and endothelium, within an extracellular matrix (ECM) substrate closely resembling that of native lung. To generate the tissues, rat lungs are inflated with agarose, sliced into 450 µm-thick slices, cut into strips, and decellularized. The resulting acellular ECM scaffolds are then reseeded with primary endothelial cells, fibroblasts, and alveolar epithelial type 2 cells (AEC2s). AEC2s can be maintained in ELT culture for at least 7 days with a serum-free, chemically-defined growth medium. Throughout the tissue preparation and culture process, the slices are clipped into a cassette system that facilitates handling and standardized cell seeding of multiple ELTs in parallel. These ELTs represent an organotypic culture platform that should facilitate investigations of cell-cell and cell-matrix interactions within the alveolus as well as biochemical signals regulating AEC2s and their niche.

Diabetes doubles the risk of developing heart failure (HF). As the prevalence of diabetes grows, so will HF unless the mechanisms connecting these diseases can be identified. Methylglyoxal (MG) is a glycolysis by-product that forms irreversible modifications on lysine and arginine, called glycation. We previously found that myofilament MG glycation causes sarcomere contractile dysfunction and is increased in patients with diabetes and HF. The aim of this study was to discover the molecular mechanisms by which MG glycation of myofilament proteins cause sarcomere dysfunction and to identify therapeutic avenues to compensate. In humans with type 2 diabetes without HF, we found increased glycation of sarcomeric actin compared to non-diabetics and it correlated with decreased calcium sensitivity. Depressed calcium sensitivity is pathogenic for HF, therefore myofilament glycation represents a promising therapeutic target to inhibit the development of HF in diabetics. To identify possible therapeutic targets, we further defined the molecular actions of myofilament glycation. Skinned myocytes exposed to 100 μM MG exhibited decreased calcium sensitivity, maximal calcium-activated force, and crossbridge kinetics. Replicating MG's functional affects using a computer simulation of sarcomere function predicted simultaneous decreases in tropomyosin's blocked-to-closed rate transition and crossbridge duty cycle were consistent with all experimental findings. Stopped-flow experiments and ATPase activity confirmed MG decreased the blocked-to-closed transition rate. Currently, no therapeutics target tropomyosin, so as proof-of-principal, we used a n-terminal peptide of myosin-binding protein C, previously shown to alter tropomyosin's position on actin. C0C2 completely rescued MG-induced calcium desensitization, suggesting a possible treatment for diabetic HF., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Paradoxically, many microRNAs appear to exhibit entirely opposite functions when placed in different contexts. For example, miR-125b has been shown to be pro-apoptotic in some studies, but anti-apoptotic in others. To investigate this phenomenon, we combine computational modeling with experimental approaches to examine how the function of miR-125b in apoptosis varies with respect to the expression levels of its pro-apoptotic and anti-apoptotic targets. In doing so, we elucidate a general trend that miR-125b is more pro-apoptotic when its anti-apoptotic targets are overexpressed, whereas it is more anti-apoptotic when its pro-apoptotic targets are overexpressed. We show that it is possible to completely reverse miR-125b's function in apoptosis by modifying the expression levels of its target genes. Furthermore, miR-125b's function may also be altered by the presence of anticancer drugs. These results suggest that the function of a microRNA can vary substantially and is dependent on its target gene expression levels., Competing Interests: The authors declare no competing interests., (© 2021 The Authors.)