A Paradigm Shift in Assessment of Scientific Skills in Undergraduate Medical Education.

The General Medical Council's publication 'Outcomes for Graduates' places emphasis on doctors being able to integrate biomedical science, research and scholarship with clinical practice. In response, a new paradigm of assessment was introduced for the intercalated Bachelor of Science program at Imperial College School of Medicine in 2019. This innovative approach involves authentic "active learning" assessments analogous to tasks encountered in a research environment and intends to test a wider range of applied scientific skills than traditional examinations. Written assessments include a "Letter to the Editor", scientific abstract, and production of a lay summary. A clinical case study titled "Science in Context" presents a real or virtual patient, with evaluation of current and emerging evidence within that field. Another assessment emulates the academic publishing process: groups submit a literature review and engage in reciprocal peer review of another group's work. A rebuttal letter accompanies the final submission, detailing how feedback was addressed. Scientific presentation skills are developed through tasks including a research proposal pitch, discussion of therapies or diagnostics, or review of a paper. A data management assignment develops skills in hypothesis generation, performing analysis, and drawing conclusions. Finally, students conduct an original research project which is assessed via a written report in the format of a research paper and an oral presentation involving critical analysis of their project. We aspire to train clinicians who apply scientific principles to critique the evidence base of medical practice and possess the skillset to conduct high-quality research underpinned by the principles of best clinical and academic practice. Assessment drives learning, and active learning has been demonstrated to enhance academic performance and reduce attainment gaps in science education. We therefore believe this strategy will help to successfully shape our students as future scientists and scholars as well as clinical practitioners and professionals.

Development a flexible light-sheet fluorescence microscope for high-speed 3D imaging of calcium dynamics and 3D imaging of cellular microstructure.

We report a flexible light-sheet fluorescence microscope (LSFM) designed for studying dynamic events in cardiac tissue at high speed in 3D and the correlation of these events to cell microstructure. The system employs two illumination-detection modes: the first uses angle-dithering of a Gaussian light sheet combined with remote refocusing of the detection plane for video-rate volumetric imaging; the second combines digitally-scanned light-sheet illumination with an axially-swept light-sheet waist and stage-scanned acquisition for improved axial resolution compared to the first mode. We present a characterisation of the spatial resolution of the system in both modes. The first illumination-detection mode achieves dual spectral-channel imaging at 25 volumes per second with 1024 × 200 × 50 voxel volumes and is demonstrated by time-lapse imaging of calcium dynamics in a live cardiomyocyte. The second illumination-detection mode is demonstrated through the acquisition of a higher spatial resolution structural map of the t-tubule network in a fixed cardiomyocyte cell.

Recent advances in understanding cardiac contractility in health and disease.

The aim of this review is to provide the reader with a synopsis of some of the emerging ideas and experimental findings in cardiac physiology and pathophysiology that were published in 2015. To provide context for the non-specialist, a brief summary of cardiac contraction and calcium (Ca) regulation in the heart in health and disease is provided. Thereafter, some recently published articles are introduced that indicate the current thinking on (1) the Ca regulatory pathways modulated by Ca/calmodulin-dependent protein kinase II, (2) the potential influences of nitrosylation by nitric oxide or S-nitrosated proteins, (3) newly observed effects of reactive oxygen species (ROS) on contraction and Ca regulation following myocardial infarction and a possible link with changes in mitochondrial Ca, and (4) the effects of some of these signaling pathways on late Na current and pro-arrhythmic afterdepolarizations as well as the effects of transverse tubule disturbances.

Non-linear optical microscopy sheds light on cardiovascular disease.

Many cardiac diseases have been associated with increased fibrosis and changes in the organization of fibrillar collagen. The degree of fibrosis is routinely analyzed with invasive histological and immunohistochemical methods, giving a limited and qualitative understanding of the tissue's morphological adaptation to disease. Our aim is to quantitatively evaluate the increase in fibrosis by three-dimensional imaging of the collagen network in the myocardium using the non-linear optical microscopy techniques Two-Photon Excitation microscopy (TPE) and Second Harmonic signal Generation (SHG). No sample staining is needed because numerous endogenous fluorophores are excited by a two-photon mechanism and highly non-centrosymmetric structures such as collagen generate strong second harmonic signals. We propose for the first time a 3D quantitative analysis to carefully evaluate the increased fibrosis in tissue from a rat model of heart failure post myocardial infarction. We show how to measure changes in fibrosis from the backward SHG (B(SHG)) alone, as only backward-propagating SHG is accessible for true in vivo applications. A 5-fold increase in collagen I fibrosis is detected in the remote surviving myocardium measured 20 weeks after infarction. The spatial distribution is also shown to change markedly, providing insight into the morphology of disease progression.

Plasticity of surface structures and ß(2)-adrenergic receptor localization in failing ventricular cardiomyocytes during recovery from heart failure.

BACKGROUND: Cardiomyocyte surface morphology and T-tubular structure are significantly disrupted in chronic heart failure, with important functional sequelae, including redistribution of sarcolemmal ß(2)-adrenergic receptors (ß(2)AR) and localized secondary messenger signaling. Plasticity of these changes in the reverse remodeled failing ventricle is unknown. We used AAV9.SERCA2a gene therapy to rescue failing rat hearts and measured z-groove index, T-tubule density, and compartmentalized ß(2)AR-mediated cAMP signals, using a combined nanoscale scanning ion conductance microscopy-Förster resonance energy transfer technique. METHODS AND RESULTS: Cardiomyocyte surface morphology, quantified by z-groove index and T-tubule density, was normalized in reverse-remodeled hearts after SERCA2a gene therapy. Recovery of sarcolemmal microstructure correlated with functional ß(2)AR redistribution back into the z-groove and T-tubular network, whereas minimal cAMP responses were initiated after local ß(2)AR stimulation of crest membrane, as observed in failing cardiomyocytes. Improvement of ß(2)AR localization was associated with recovery of ßAR-stimulated contractile responses in rescued cardiomyocytes. Retubulation was associated with reduced spatial heterogeneity of electrically stimulated calcium transients and recovery of myocardial BIN-1 and TCAP protein expression but not junctophilin-2. CONCLUSIONS: In summary, abnormalities of sarcolemmal structure in heart failure show plasticity with reappearance of z-grooves and T-tubules in reverse-remodeled hearts. Recovery of surface topology is necessary for normalization of ß(2)AR location and signaling responses.

High-speed 2D and 3D fluorescence microscopy of cardiac myocytes.

Oblique plane microscopy (OPM) is a light sheet microscopy technique that uses a single high numerical aperture microscope objective to both illuminate a tilted plane within the specimen and to obtain an image of the tilted illuminated plane. In this paper, we present a new OPM configuration that enables both the illumination and detection focal planes to be swept simultaneously and remotely through the sample volume, enabling high speed volumetric imaging. We demonstrate the high speed imaging capabilities of the system by imaging calcium dynamics in cardiac myocytes in 2D at 926 frames per second and in 3D at 21 volumes per second. In the future, higher frame rate CCD cameras will enable volumetric imaging at much greater rates, leading to new capabilities to study dynamic events in cells at high speeds in two and three dimensions.

SERCA2a gene transfer decreases sarcoplasmic reticulum calcium leak and reduces ventricular arrhythmias in a model of chronic heart failure.

BACKGROUND: Sarcoplasmic reticulum calcium ATPase 2a (SERCA2a) gene therapy improves mechanical function in heart failure and is under evaluation in a clinical trial. A critical question is whether SERCA2a gene therapy predisposes to increased sarcoplasmic reticulum calcium (SR Ca(2+)) leak, cellular triggered activity, and ventricular arrhythmias in the failing heart. METHODS AND RESULTS: We studied the influence of SERCA2a gene therapy on ventricular arrhythmogenesis in a rat chronic heart failure model. ECG telemetry studies revealed a significant antiarrhythmic effect of SERCA2a gene therapy with reduction of both spontaneous and catecholamine-induced arrhythmias in vivo. SERCA2a gene therapy also reduced susceptibility to reentry arrhythmias in ex vivo programmed electrical stimulation studies. Subcellular Ca(2+) homeostasis and spontaneous SR Ca(2+) leak characteristics were measured in failing cardiomyocytes transfected in vivo with a novel AAV9.SERCA2a vector. SR Ca(2+) leak was reduced after SERCA2a gene therapy, with reversal of the greater spark mass observed in the failing myocytes, despite normalization of SR Ca(2+) load. SERCA2a reduced ryanodine receptor phosphorylation, thereby resetting SR Ca(2+) leak threshold, leading to reduced triggered activity in vitro. Both indirect effects of reverse remodeling and direct SERCA2a effects appear to underlie the antiarrhythmic action. CONCLUSIONS: SERCA2a gene therapy stabilizes SR Ca(2+) load, reduces ryanodine receptor phosphorylation and decreases SR Ca(2+) leak, and reduces cellular triggered activity in vitro and spontaneous and catecholamine-induced ventricular arrhythmias in vivo in failing hearts. SERCA2a gene therapy did not therefore predispose to arrhythmias and may represent a novel antiarrhythmic strategy in heart failure.

Loss of T-tubules and other changes to surface topography in ventricular myocytes from failing human and rat heart.

T-tubular invaginations of the sarcolemma of ventricular cardiomyocytes contain junctional structures functionally coupling L-type calcium channels to the sarcoplasmic reticulum calcium-release channels (the ryanodine receptors), and therefore their configuration controls the gain of calcium-induced calcium release (CICR). Studies primarily in rodent myocardium have shown the importance of T-tubular structures for calcium transient kinetics and have linked T-tubule disruption to delayed CICR. However, there is disagreement as to the nature of T-tubule changes in human heart failure. We studied isolated ventricular myocytes from patients with ischemic heart disease, idiopathic dilated cardiomyopathy, and hypertrophic obstructive cardiomyopathy and determined T-tubule structure with either the fluorescent membrane dye di-8-ANNEPs or the scanning ion conductance microscope (SICM). The SICM uses a scanning pipette to produce a topographic representation of the surface of the live cell by a non-optical method. We have also compared ventricular myocytes from a rat model of chronic heart failure after myocardial infarction. T-tubule loss, shown by both ANNEPs staining and SICM imaging, was pronounced in human myocytes from all etiologies of disease. SICM imaging showed additional changes in surface structure, with flattening and loss of Z-groove definition common to all etiologies. Rat myocytes from the chronic heart failure model also showed both T-tubule and Z-groove loss, as well as increased spark frequency and greater spark amplitude. This study confirms the loss of T-tubules as part of the phenotypic change in the failing human myocyte, but it also shows that this is part of a wider spectrum of alterations in surface morphology.

Effects of epidural-and-general anesthesia combined versus general anesthesia alone on the venous hemodynamics of the lower limb. A randomized study.

Our hypothesis was that, due to its sympatholytic action, epidural anesthesia (EA) administered as part of anesthesia in abdominal surgery would generate a marked venous leg flow enhancement, thus aiding in the prevention of peroperative venous stasis. We studied, and comprehensively quantified the venous haemodynamic changes in the lower limb during and immediately after abdominal surgery performed under EA and general (GA) anesthesia combined, in comparison to GA alone. This is a prospective, randomized, controlled study, stratified for hypertension and smoking, comprising ASA 1-2 patients undergoing elective total abdominal hysterectomy. Those with peripheral vascular or chronic venous disease, prior DVT or BMI>35 were excluded. Eligible recruits received either GA (Group GA) (n = 10; age 36-65, median 50) alone or epidural anesthesia (EA) and GA combined (Group EA/GA) (n = 9; age 32-58, median 46). EA (L(1-2)) was administered using lignocaine 2%. Both groups had GA induced with fentanyl and propofol, maintained with N(2)O and isoflurane; larygoscopy was facilitated with vecuronium; analgesia was provided either with morphine (Group GA) or epidurally with 2% lignocaine boli (Group EA/GA). Hemodynamics were determined at the popliteal vein in the horizontal supine position at baseline (resting prior to anesthesia), post epidural (20 min after delivery of EA), post induction (15 min after laryngeal intubation), surgery (upon uterus removal) and recovery (30 min after extubation). There was no difference in the mean velocity[V(mean)] between the 2 groups at baseline (p = 0.35([Mann-Whitney])), and post induction (p = 0.5([Mann-Whitney])). However V(mean) was significantly higher in Group EA/GA than Group GA, both at surgery (point estimate[PE]: 1.8 cm/s; 95% CI: 0.01, 6.3 cm/s; p <0.05([Mann-Whitney])) and recovery (PE: 2.6 cm/s; 95% CI: 0.4, 5.1 cm/s; p = 0.02([Mann-Whitney])). Volume flow[V(Q)] was similar in the 2 groups at baseline and post induction (both, p >0.1([Mann-Whitney])), but was significantly higher in Group EA/GA at surgery (PE: 54 ml/min; 95% CI: 18, 159 ml/min; p = 0.045([Mann-Whitney])) and recovery (PE: 49 ml/min; 95% CI: 16, 129 ml/min; p=0.0037([Mann-Whitney])). Peak velocity, V(mean) and V(Q) increased significantly post epidural in Group EA/GA. Contrary to the venous leg flow attenuation in elective abdominal surgery under GA and upon its recovery, EA administered as part of GA is associated with a significant enhancement of both V(mean) and V(Q). This beneficial hemodynamic effect of EA at the vulnerable stage of recovery may be critically essential in light of enhanced blood viscosity, fibrinolytic shut-down, endothelial/platelet activation and immobility, acting in synergy with putative cardiorespiratory protection. The results of this study lend support to the preferential selection of combined EA/GA in subjects at high risk for venous thromboembolism, particularly when optimal DVT prophylaxis is practically unattainable due to limitations pertaining to the nature of surgery.