Bidirectional epithelial-mesenchymal crosstalk provides self-sustaining profibrotic signals in pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is the prototypic progressive fibrotic lung disease with a median survival of 2 to 4 years. Injury to and/or dysfunction of the alveolar epithelium is strongly implicated in IPF disease initiation, but the factors that determine whether fibrosis progresses rather than normal tissue repair occurs remain poorly understood. We previously demonstrated that zinc finger E-box-binding homeobox 1-mediated epithelial-mesenchymal transition in human alveolar epithelial type II (ATII) cells augments transforming growth factor-ß-induced profibrogenic responses in underlying lung fibroblasts via paracrine signaling. Here, we investigated bidirectional epithelial-mesenchymal crosstalk and its potential to drive fibrosis progression. RNA-Seq of lung fibroblasts exposed to conditioned media from ATII cells undergoing RAS-induced epithelial-mesenchymal transition identified many differentially expressed genes including those involved in cell migration and extracellular matrix regulation. We confirmed that paracrine signaling between RAS-activated ATII cells and fibroblasts augmented fibroblast recruitment and demonstrated that this involved a zinc finger E-box-binding homeobox 1-tissue plasminogen activator axis. In a reciprocal fashion, paracrine signaling from transforming growth factor-ß-activated lung fibroblasts or IPF fibroblasts induced RAS activation in ATII cells, at least partially through the secreted protein acidic and rich in cysteine, which may signal via the epithelial growth factor receptor via epithelial growth factor-like repeats. Together, these data identify that aberrant bidirectional epithelial-mesenchymal crosstalk in IPF drives a chronic feedback loop that maintains a wound-healing phenotype and provides self-sustaining profibrotic signals.

No relevant pharmacokinetic drug-drug interaction between nintedanib and pirfenidone.

Nintedanib and pirfenidone are approved treatments for idiopathic pulmonary fibrosis (IPF). This open-label, two-group trial investigated the pharmacokinetic drug-drug interaction between these two drugs in patients with IPF.Subjects not treated with antifibrotics at screening (group 1, n=20) received a single nintedanib dose (150 mg) followed by pirfenidone (titrated to 801 mg thrice daily) for 3 weeks, with a further single nintedanib dose (150 mg) on the last day (day 23). Subjects treated with pirfenidone at screening (group 2, n=17) continued to receive pirfenidone alone (801 mg thrice daily) for 7 days, then co-administered with nintedanib (150 mg twice daily) for a further 7 days, before single doses of both treatments on day 16.In group 1, adjusted geometric mean (gMean) ratios (with/without pirfenidone) were 88.6% and 80.6% for nintedanib area under the plasma concentration-time curve (AUC) and maximum plasma concentration (Cmax), respectively. In group 2, gMean ratios (with/without nintedanib) were 97.2% and 99.5% for pirfenidone AUC and Cmax, respectively. For all parameters, the 90% confidence intervals included 100%, suggesting similar exposure for administration alone and when co-administered. Both treatments were well tolerated.These data indicate there is no relevant pharmacokinetic drug-drug interaction between nintedanib and pirfenidone when co-administered in IPF patients.

"Velcro-type" crackles predict specific radiologic features of fibrotic interstitial lung disease.

BACKGROUND: "Velcro-type" crackles on chest auscultation are considered a typical acoustic finding of Fibrotic Interstitial Lung Disease (FILD), however whether they may have a role in the early detection of these disorders has been unknown. This study investigated how "Velcro-type" crackles correlate with the presence of distinct patterns of FILD and individual radiologic features of pulmonary fibrosis on High Resolution Computed Tomography (HRCT). METHODS: Lung sounds were digitally recorded from subjects immediately prior to undergoing clinically indicated chest HRCT. Audio files were independently assessed by two chest physicians and both full volume and single HRCT sections corresponding to the recording sites were extracted. The relationships between audible "Velcro-type" crackles and radiologic HRCT patterns and individual features of pulmonary fibrosis were investigated using multivariate regression models. RESULTS: 148 subjects were enrolled: bilateral "Velcro-type" crackles predicted the presence of FILD at HRCT (OR 13.46, 95% CI 5.85-30.96, p < 0.001) and most strongly the Usual Interstitial Pneumonia (UIP) pattern (OR 19.8, 95% CI 5.28-74.25, p < 0.001). Extent of isolated reticulation (OR 2.04, 95% CI 1.62-2.57, p < 0.001), honeycombing (OR 1.88, 95% CI 1.24-2.83, < 0.01), ground glass opacities (OR 1.74, 95% CI 1.29-2.32, p < 0.001) and traction bronchiectasis (OR 1.55, 95% CI 1.03-2.32, p < 0.05) were all independently associated with the presence of "Velcro-type" crackles. CONCLUSIONS: "Velcro-type" crackles predict the presence of FILD and directly correlate with the extent of distinct radiologic features of pulmonary fibrosis. Such evidence provides grounds for further investigation of lung sounds as an early identification tool in FILD.

Functional Magnetic Resonance Imaging with Concurrent Urodynamic Testing Identifies Brain Structures Involved in Micturition Cycle in Patients with Multiple Sclerosis.

PURPOSE: Neurogenic lower urinary tract dysfunction, which is common in patients with multiple sclerosis, has a significant impact on quality of life. In this study we sought to determine brain activity processes during the micturition cycle in female patients with multiple sclerosis and neurogenic lower urinary tract dysfunction. MATERIALS AND METHODS: We report brain activity on functional magnetic resonance imaging and simultaneous urodynamic testing in 23 ambulatory female patients with multiple sclerosis. Individual functional magnetic resonance imaging activation maps at strong desire to void and at initiation of voiding were calculated and averaged at Montreal Neuroimaging Institute. Areas of significant activation were identified in these average maps. Subgroup analysis was performed in patients with elicitable neurogenic detrusor overactivity or detrusor-sphincter dyssynergia. RESULTS: Group analysis of all patients at strong desire to void yielded areas of activation in regions associated with executive function (frontal gyrus), emotional regulation (cingulate gyrus) and motor control (putamen, cerebellum and precuneus). Comparison of the average change in activation between previously reported healthy controls and patients with multiple sclerosis showed predominantly stronger, more focal activation in the former and lower, more diffused activation in the latter. Patients with multiple sclerosis who had demonstrable neurogenic detrusor overactivity and detrusor-sphincter dyssynergia showed a trend toward distinct brain activation at full urge and at initiation of voiding respectively. CONCLUSIONS: We successfully studied brain activation during the entire micturition cycle in female patients with neurogenic lower urinary tract dysfunction and multiple sclerosis using a concurrent functional magnetic resonance imaging/urodynamic testing platform. Understanding the central neural processes involved in specific parts of micturition in patients with neurogenic lower urinary tract dysfunction may identify areas of interest for future intervention.

Rural health professionals' experiences in implementing advance care planning: a focus group study.

Advance care planning (ACP) is described as an ongoing discussion between a patient, their family and healthcare professionals (HCPs) to understand a patient's wishes for future health care. Legislation supporting ACP in Western Australia is relatively new and HCPs are still learning about the process and implementation. This study aimed to provide a rich description of rural health professionals' perceptions and experiences with ACP within the context of their professional role and to identify systemic issues and training needs. Ten focus groups were conducted throughout 2014 with a total of 55 rural participants including general practitioners (n=15), general practice registrars (n=6), practice nurses (n=18), community nurses (n=4) and hospital nurses (n=12) in the south-western regions of Western Australia. Thematic analysis has identified the following themes regarding ACP: benefits to patients and families; professional roles in ACP; barriers and enablers; and systems for communicating ACP. HCPs have self-determined their roles in the ACP process, which currently leaves some components of the process unaccounted for, suggesting that collaboration between HCPs working together in a rural health setting and a standardised system for distributing these documents may assist with the implementation of ACP.

Functional magnetic resonance imaging during urodynamic testing identifies brain structures initiating micturition.

PURPOSE: Normal voiding in neurologically intact patients is triggered by the release of tonic inhibition from suprapontine centers, allowing the pontine micturition center to trigger the voiding reflex. Supraspinal mechanisms of voluntary voiding in humans are just beginning to be described via functional neuroimaging. We further elucidated brain activity processes during voiding using functional magnetic resonance imaging in normal females to gain better understanding of normal voiding as well as changes that may occur in voiding dysfunction. MATERIALS AND METHODS: We screened 13 healthy premenopausal female volunteers using baseline clinic urodynamics to document normal voiding parameters. We then recorded brain activity via functional magnetic resonance imaging and simultaneous urodynamics, including the pressure flow voiding phase. After motion correction of functional magnetic resonance images we performed activation and connectivity analyses in 10 subjects. RESULTS: Group analysis revealed consistent activation areas, including regions for motor control (cerebellum, thalamus, caudate, lentiform nucleus, red nucleus, supplementary motor area and post-central gyrus), emotion (anterior/posterior cingulate gyrus and insula), executive function (left superior frontal gyrus) and a focal region in the pons. Connectivity analysis demonstrated strong interconnectivity of the pontine micturition center with many short-range and long-range cortical clusters. CONCLUSIONS: Our study is one of the first reports of brain activation centers associated with micturition initiation in normal healthy females. Results show activation of a brain network consisting of regions for motor control, executive function and emotion processing. Further studies are planned to create and validate a model of brain activity during normal voiding in women.

Renal deterioration in multiple sclerosis patients with neurovesical dysfunction.

OBJECTIVES: Our aims were to determine the prevalence of renal ultrasound (RUS) abnormalities over time in multiple sclerosis (MS) patients with lower urinary tract symptoms (LUTS). METHODS: Data were examined retrospectively from MS patients with LUTS, from 2000-2009. Study inclusion requirements were both baseline urodynamics (UD) and RUS data, with followup RUS at ≥ 12 months. Age, time since diagnosis (TSD), MS subtype and the UD/RUS results were evaluated for associations. RESULTS: At presentation, 173 subjects underwent UD and RUS, but only 89 had a repeat RUS at ≥ 12 months. Median followup was 61 months. Initial RUS abnormalities were found in 10 (5.8%) subjects. At followup, upper urinary tract (UUT) abnormalities were seen in 11 (12.4%) subjects. Patients > 49 years old were more likely to have an abnormality (OR 0.181, 95% CI 0.037-0.892, p = 0.04). Patients with abnormal compliance were also more likely to have an abnormal followup RUS (OR 0.185, 95% CI 0.037-0.924, p = 0.04). No other demographic or UD factor was associated with RUS abnormalities. CONCLUSIONS: The development of structural UUT changes is low in MS patients. Urodynamic studies are useful for LUTS treatment strategies in complicated patients, but UD does not appear to have much impact with regard to upper tract changes.

Idiopathic pulmonary fibrosis: recent trials and current drug therapy.

Idiopathic pulmonary fibrosis (IPF) is a chronic fibrotic lung disease with increasing incidence; the median survival is only 35 months and as yet no therapy has been proven to prolong survival. Recent unexpected randomised controlled trial (RCT) results and the conflicting evaluations of drug efficacy by regulatory agencies when considering the approval of pirfenidone have emphasised that we remain in the first stages of both our understanding of disease-relevant therapeutic targets and in our ability to investigate these putative targets with well-designed RCT. Three phase III trials are, however, anticipated to report results in 2014 and there is cautious optimism that we may be entering an era of mechanism-based anti-fibrotic therapies proven by large RCT to modify disease progression. We must now address how to practically translate these therapies safely and with maximal efficacy from a homogenous RCT population to the 'real-life' heterogeneous population of patients with IPF. The role of a formal multi-disciplinary team meeting in a specialist centre with expertise in IPF is key to this. New methodological and ethical research challenges will arise as we enter an era of potential combination therapy; standardized, robust RCT design will be central to meeting these challenges if we are to enable ongoing progress in our aim of increasing both the length and quality of life of patients with IPF.

Rituximab versus intravenous cyclophosphamide in patients with connective tissue disease-associated interstitial lung disease in the UK (RECITAL): a double-blind, double-dummy, randomised, controlled, phase 2b trial.

BACKGROUND: Rituximab is often used as rescue therapy in interstitial lung disease (ILD) associated with connective tissue disease (CTD), but has not been studied in clinical trials. This study aimed to assess whether rituximab is superior to cyclophosphamide as a treatment for severe or progressive CTD associated ILD. METHODS: We conducted a randomised, double-blind, double-dummy, phase 2b trial to assess the superiority of rituximab compared with cyclophosphamide. Patients aged 18-80 years with severe or progressive ILD related to scleroderma, idiopathic inflammatory myositis, or mixed CTD, recruited across 11 specialist ILD or rheumatology centres in the UK, were randomly assigned (1:1) to receive rituximab (1000 mg at weeks 0 and 2 intravenously) or cyclophosphamide (600 mg/m2 body surface area every 4 weeks intravenously for six doses). The primary endpoint was rate of change in forced vital capacity (FVC) at 24 weeks compared with baseline, analysed using a mixed-effects model with random intercepts, adjusted for baseline FVC and CTD type. Prespecified secondary endpoints reported in this Article were change in FVC at 48 weeks versus baseline; changes from baseline in 6 min walk distance, diffusing capacity of the lung for carbon monoxide (DLCO), physician-assessed global disease activity (GDA) score, and quality-of-life scores on the St George's Respiratory Questionnaire (SGRQ), King's Brief Interstitial Lung Disease (KBILD) questionnaire, and European Quality of Life Five-Dimension (EQ-5D) questionnaire at 24 and 48 weeks; overall survival, progression-free survival, and time to treatment failure; and corticosteroid use. All endpoints were analysed in the modified intention-to-treat population, which comprised all patients who received at least one dose of study drug. This trial is registered with ClinicalTrials.gov (NCT01862926). FINDINGS: Between Dec 1, 2014, and March 31, 2020, we screened 145 participants, of whom 101 participants were randomly allocated: 50 (50%) to receive cyclophosphamide and 51 (50%) to receive rituximab. 48 (96%) participants in the cyclophosphamide group and 49 (96%) in the rituximab group received at least one dose of treatment and were included in analyses; 43 (86%) participants in the cyclophosphamide group and 42 (82%) participants in the rituximab group completed 24 weeks of treatment and follow-up. At 24 weeks, FVC was improved from baseline in both the cyclophosphamide group (unadjusted mean increase 99 mL [SD 329]) and the rituximab group (97 mL [234]); in the adjusted mixed-effects model, the difference in the primary endpoint at 24 weeks was -40 mL (95% CI -153 to 74; p=0·49) between the rituximab group and the cyclophosphamide group. KBILD quality-of-life scores were improved at 24 weeks by a mean 9·4 points (SD 20·8) in the cyclophosphamide group and 8·8 points (17·0) in the rituximab group. No significant differences in secondary endpoints were identified between the treatment groups, with the exception of change in GDA score at week 48, which favoured cyclophosphamide (difference 0·90 [95% CI 0·11 to 1·68]). Improvements in lung function and respiratory-related quality-of-life measures were observed in both treatment groups. Lower corticosteroid exposure over 48 weeks of follow-up was recorded in the rituximab group. Two (4%) of 48 participants who received cyclophosphamide and three (6%) of 49 who received rituximab died during the study, all due to complications of CTD or ILD. Overall survival, progression-free survival, and time to treatment failure did not significantly differ between the two groups. All participants reported at least one adverse event during the study. Numerically fewer adverse events were reported by participants receiving rituximab (445 events) than those receiving cyclophosphamide (646 events). Gastrointestinal and respiratory disorders were the most commonly reported adverse events in both groups. There were 62 serious adverse events of which 33 occurred in the cyclophosphamide group and 29 in the rituximab group. INTERPRETATION: Rituximab was not superior to cyclophosphamide to treat patients with CTD-ILD, although participants in both treatment groups had increased FVC at 24 weeks, in addition to clinically important improvements in patient-reported quality of life. Rituximab was associated with fewer adverse events. Rituximab should be considered as a therapeutic alternative to cyclophosphamide in individuals with CTD-ILD requiring intravenous therapy. FUNDING: Efficacy and Mechanism Evaluation Programme (Medical Research Council and National Institute for Health Research, UK).

Individualised Exercise Training Enhances Antioxidant Buffering Capacity in Idiopathic Pulmonary Fibrosis.

Exercise training is recommended for patients with idiopathic pulmonary fibrosis (IPF); however, the mechanism(s) underlying its physiological benefits remain unclear. We investigated the effects of an individualised aerobic interval training programme on exercise capacity and redox status in IPF patients. IPF patients were recruited prospectively to an 8-week, twice-weekly cardiopulmonary exercise test (CPET)-derived structured responsive exercise training programme (SRETP). Systemic redox status was assessed pre- and post-CPET at baseline and following SRETP completion. An age- and sex-matched non-IPF control cohort was recruited for baseline comparison only. At baseline, IPF patients (n = 15) had evidence of increased oxidative stress compared with the controls as judged by; the plasma reduced/oxidised glutathione ratio (median, control 1856 vs. IPF 736 p = 0.046). Eleven IPF patients completed the SRETP (median adherence 88%). Following SRETP completion, there was a significant improvement in exercise capacity assessed via the constant work-rate endurance time (+82%, p = 0.003). This was accompanied by an improvement in post-exercise redox status (in favour of antioxidants) assessed via serum total free thiols (median increase, +0.26 µmol/g protein p = 0.005) and total glutathione concentration (+0.73 µM p = 0.03), as well as a decrease in post-exercise lipid peroxidation products (-1.20 µM p = 0.02). Following SRETP completion, post-exercise circulating nitrite concentrations were significantly lower compared with baseline (-0.39 µM p = 0.04), suggestive of exercise-induced nitrite utilisation. The SRETP increased both endurance time and systemic antioxidant capacity in IPF patients. The observed reduction in nitrite concentrations provides a mechanistic rationale to investigate nitrite/nitrate supplementation in IPF patients.

A hyperinflammation clinical risk tool, HI5-NEWS2, stratifies hospitalised COVID-19 patients to associate risk of death and effect of early dexamethasone in an observational cohort.

BACKGROUND: The success of early dexamethasone therapy for hospitalised COVID-19 cases in treatment of Sars-CoV-2 infection may predominantly reflect its anti-inflammatory action against a hyperinflammation (HI) response. It is likely that there is substantial heterogeneity in HI responses in COVID-19. METHODS: Blood CRP, ferritin, neutrophil, lymphocyte and platelet counts were scored to assess HI (HI5) and combined with a validated measure of generalised medical deterioration (NEWS2) before day 2. Our primary outcome was 28 day mortality from early treatment with dexamethasone stratified by HI5-NEWS2 status. FINDINGS: Of 1265 patients, high risk of HI (high HI5-NEWS2) (n = 367, 29.0%) conferred a strikingly increased mortality (36.0% vs 7.8%; Age adjusted hazard ratio (aHR) 5.9; 95% CI 3.6-9.8, p<0.001) compared to the low risk group (n = 455, 36.0%). An intermediate risk group (n = 443, 35.0%) also showed significantly higher mortality than the low risk group (17.6% vs 7.8%), aHR 2.2, p = 0.005). Early dexamethasone treatment conferred a 50.0% reduction in mortality in the high risk group (36.0% to 18.0%, aHR 0.56, p = 0.007). The intermediate risk group showed a trend to reduction in mortality (17.8% to 10.3%, aHR 0.82, p = 0.46) which was not observed in the low risk group (7.8% to 9.2%, aHR 1.4, p = 0.31). INTERPRETATION: Higher HI5-NEWS2 scores measured at COVID-19 diagnosis, strongly associate with increased mortality at 28 days. Significant reduction in mortality with early dexamethasone treatment was only observed in the high risk group. Therefore, the HI5-NEWS2 score could be utilised to stratify randomised clinical trials to test whether intensified anti-inflammatory therapy would further benefit high risk patients and whether alternative approaches would benefit low risk groups. Considering its recognised morbidity, we suggest that early dexamethasone should not be routinely prescribed for HI5-NEWS2 low risk individuals with COVID-19 and clinicians should cautiously assess the risk benefit of this intervention in all cases.

Spatially resolved deconvolution of the fibrotic niche in lung fibrosis.

A defining pathological feature of human lung fibrosis is localized tissue heterogeneity, which challenges the interpretation of transcriptomic studies that typically lose spatial information. Here we investigate spatial gene expression in diagnostic tissue using digital profiling technology. We identify distinct, region-specific gene expression signatures as well as shared gene signatures. By integration with single-cell data, we spatially map the cellular composition within and distant from the fibrotic niche, demonstrating discrete changes in homeostatic and pathologic cell populations even in morphologically preserved lung, while through ligand-receptor analysis, we investigate cellular cross-talk within the fibrotic niche. We confirm findings through bioinformatic, tissue, and in vitro analyses, identifying that loss of NFKB inhibitor zeta in alveolar epithelial cells dysregulates the TGFß/IL-6 signaling axis, which may impair homeostatic responses to environmental stress. Thus, spatially resolved deconvolution advances understanding of cell composition and microenvironment in human lung fibrogenesis.

Pseudohypoxic HIF pathway activation dysregulates collagen structure-function in human lung fibrosis.

Extracellular matrix (ECM) stiffening with downstream activation of mechanosensitive pathways is strongly implicated in fibrosis. We previously reported that altered collagen nanoarchitecture is a key determinant of pathogenetic ECM structure-function in human fibrosis (Jones et al., 2018). Here, through human tissue, bioinformatic and ex vivo studies we provide evidence that hypoxia-inducible factor (HIF) pathway activation is a critical pathway for this process regardless of the oxygen status (pseudohypoxia). Whilst TGFß increased the rate of fibrillar collagen synthesis, HIF pathway activation was required to dysregulate post-translational modification of fibrillar collagen, promoting pyridinoline cross-linking, altering collagen nanostructure, and increasing tissue stiffness. In vitro, knockdown of Factor Inhibiting HIF (FIH), which modulates HIF activity, or oxidative stress caused pseudohypoxic HIF activation in the normal fibroblasts. By contrast, endogenous FIH activity was reduced in fibroblasts from patients with lung fibrosis in association with significantly increased normoxic HIF pathway activation. In human lung fibrosis tissue, HIF-mediated signalling was increased at sites of active fibrogenesis whilst subpopulations of human lung fibrosis mesenchymal cells had increases in both HIF and oxidative stress scores. Our data demonstrate that oxidative stress can drive pseudohypoxic HIF pathway activation which is a critical regulator of pathogenetic collagen structure-function in fibrosis.

Demographic and urodynamic factors associated with persistent OAB after anterior compartment prolapse repair.

INTRODUCTION: Overactive bladder (OAB) symptoms often accompany pelvic organ prolapse. While there seems to be a relationship between symptom resolution and anatomic repair, a subset of patients will not experience improvement in OAB symptoms. Our aim was to identify preoperative demographic and urodynamic (UD) parameters related to persistence of OAB symptoms after anterior vaginal prolapse (AVP) repair. METHODS: This retrospective cohort study examined demographic and UD data from patients undergoing AVP surgery. Pre- and post-operative Urogenital Distress Inventory (UDI-6) scores for frequency, urge urinary incontinence (UUI), and difficulty voiding were analyzed, as were correlations between scores and pre-operative UD data. RESULTS: From 2002 to 2008, 88 patients underwent AVP repair and were included in the final analysis. Surgery resulted in a reduction of frequency (33%), UUI (49%), and difficulty voiding (74%) at median 21 months follow-up. Change in symptom scores was unrelated to age, parity, BMI, or AVP grade, although older women reported greater improvement in difficulty emptying after repair. Improvement in difficulty emptying was related to a larger pre-operative post-void residual (PVR) (129 ml vs. 31 ml, P = 0.0008). Persistent UUI after repair was significantly related to a higher preoperative P(det)Q(max) (OR 1.056, 95% CI 1.003-1.11, P = 0.04). Other pre-operative UDS variables were not significantly related to the persistence of OAB symptoms. CONCLUSIONS: AVP repair reduces lower urinary tract symptoms (LUTS); however, 67% and 51% of patients will report persistent frequency and UUI, respectively, post-operatively. In this cohort, persistent OAB symptoms were not related to age, parity, BMI, or prolapse grade, but rather to pre-operative P(det)Q(max).

The cardiac work-loop technique: An in vitro model for identifying and profiling drug-induced changes in inotropy using rat papillary muscles.

The cardiac work-loop technique closely mimics the intrinsic in vivo movement and characteristics of cardiac muscle function. In this study, six known inotropes were profiled using the work-loop technique to evaluate the potential of this method to predict inotropy. Papillary muscles from male Sprague-Dawley rats were mounted onto an organ bath perfused with Krebs-Henseleit buffer. Following optimisation, work-loop contractions were performed that included an initial stabilisation period followed by vehicle control or drug administration. Six known inotropes were tested: digoxin, dobutamine, isoprenaline, flecainide, verapamil and atenolol. Muscle performance was evaluated by calculating power output during work-loop contraction. Digoxin, dobutamine and isoprenaline caused a significant increase in power output of muscles when compared to vehicle control. Flecainide, verapamil and atenolol significantly reduced power output of muscles. These changes in power output were reflected in alterations in work loop shapes. This is the first study in which changes in work-loop shape detailing for example the activation, shortening or passive re-lengthening have been linked to the mechanism of action of a compound. This study has demonstrated that the work-loop technique can provide an important novel method with which to assess detailed mechanisms of drug-induced effects on cardiac muscle contractility.