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AimsThe response to high frequency stimulation (HFS) is used to locate putative sites of ganglionated plexuses (GPs), which are implicated in triggering atrial fibrillation (AF). To identify topological and immunohistochemical characteristics of presumed GP sites functionally identified by HFS.Methods and resultsSixty-three atrial sites were tested with HFS in four Langendorff-perfused porcine hearts. A 3.5 mm tip quadripolar ablation catheter was used to stimulate and deliver HFS to the left and right atrial epicardium, within the local atrial refractory period. Tissue samples from sites triggering atrial ectopy/AF (ET) sites and non-ET sites were stained with choline acetyltransferase (ChAT) and tyrosine hydroxylase (TH), for quantification of parasympathetic and sympathetic nerves, respectively. The average cross-sectional area (CSA) of nerves was also calculated. Histomorphometry of six ET sites (9.5%) identified by HFS evoking at least a single atrial ectopic was compared with non-ET sites. All ET sites contained ChAT-immunoreactive (ChAT-IR) and/or TH-immunoreactive nerves (TH-IR). Nerve density was greater in ET sites compared to non-ET sites (nerves/cm2: 162.3 ± 110.9 vs. 69.65 ± 72.48; P = 0.047). Overall, TH-IR nerves had a larger CSA than ChAT-IR nerves (µm2: 11 196 ± 35 141 vs. 2070 ± 5841; P < 0.0001), but in ET sites, TH-IR nerves were smaller than in non-ET sites (µm2: 6021 ± 14 586 vs. 25 254 ± 61 499; P < 0.001).ConclusionsET sites identified by HFS contained a higher density of smaller nerves than non-ET sites. The majority of these nerves were within the atrial myocardium. This has important clinical implications for devising an effective therapeutic strategy for targeting autonomic triggers of AF.

Aims Conflicting data exist supporting differing mechanisms for sustaining ventricular fibrillation (VF), ranging from disorganised multiple-wavelet activation to organised rotational activities (RAs). Abnormal gap junction (GJ) coupling and fibrosis are important in initiation and maintenance of VF. We investigated whether differing ventricular fibrosis patterns and the degree of GJ coupling affected the underlying VF mechanism. Methods and Results Optical mapping of 65 Langendorff-perfused rat hearts was performed to study VF mechanisms in control hearts with acute GJ modulation, and separately in three differing chronic ventricular fibrosis models; compact (CF), diffuse (DiF) and patchy (PF). VF dynamics were quantified with phase mapping and frequency dominance index (FDI) analysis, a power ratio of the highest amplitude dominant frequency in the cardiac frequency spectrum. Enhanced GJ coupling with rotigaptide (n = 10) progressively organised fibrillation in a concentration-dependent manner; increasing FDI (0nM: 0.53±0.04, 80nM: 0.78±0.03, p

During viral pandemics, filtering facepiece (FFP) masks together with eye protection form the essential components of personal protective equipment (PPE) for healthcare workers. There remain concerns regarding insufficient global supply and imperfect protection offered by currently available PPE strategies. A range of full-face snorkel masks were adapted to accept high grade medical respiratory filters using bespoke-designed 3D-printed connectors. We compared the protection offered by the snorkel to that of standard PPE using a placebo-controlled respirator filtering test as well as a fluorescent droplet deposition experiment. Out of the 56 subjects tested, 42 (75%) passed filtering testing with the snorkel mask compared to 31 (55%) with a FFP3 respirator mask (p = 0.003). Amongst the 43 subjects who were not excluded following a placebo control, 85% passed filtering testing with the snorkel versus to 68% with a FFP3 mask (p = 0.008). Following front and lateral spray of fluorescence liquid particles, the snorkel mask also provided superior protection against droplet deposition within the subject's face, when compared to a standard PPE combination of FFP3 masks and eye protection (3.19x108 versus 6.81x108 fluorescence units, p0.001). The 3D printable adaptors are available for free download online at https://www.ImperialHackspace.com/COVID-19-Snorkel-Respirator-Project/.Full-face snorkel masks adapted as particulate respirators performed better than a standard PPE combination of FFP3 mask and eye protection against aerosol inhalation and droplet deposition. This adaptation is therefore a promising PPE solution for healthcare workers during highly contagious viral outbreaks.

Engineered heart tissue (EHT) strategies, by combining cells within a hydrogel matrix, may be a novel therapy for heart failure. EHTs restore cardiac function in rodent injury models, but more data are needed in clinically relevant settings. Accordingly, an upscaled EHT patch (2.5 cm × 1.5 cm × 1.5 mm) consisting of up to 20 million human induced pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) embedded in a fibrin-based hydrogel was developed. A rabbit myocardial infarction model was then established to test for feasibility and efficacy. Our data showed that hPSC-CMs in EHTs became more aligned over 28 days and had improved contraction kinetics and faster calcium transients. Blinded echocardiographic analysis revealed a significant improvement in function in infarcted hearts that received EHTs, along with reduction in infarct scar size by 35%. Vascularization from the host to the patch was observed at week 1 and stable to week 4, but electrical coupling between patch and host heart was not observed. In vivo telemetry recordings and ex vivo arrhythmia provocation protocols showed that the patch was not pro-arrhythmic. In summary, EHTs improved function and reduced scar size without causing arrhythmia, which may be due to the lack of electrical coupling between patch and host heart.

Introduction During viral pandemics, filtering facepiece (FFP) masks together with eye protection form the essential components of personal protective equipment (PPE) for healthcare workers. There remain concerns regarding insufficient global supply and imperfect protection offered by currently available PPE strategies. A range of full-face snorkel masks were adapted to accept high grade medical respiratory filters using bespoke-designed 3D-printed connectors. We compared the protection offered by the snorkel to that of standard PPE using a placebo-controlled respirator filtering test as well as a fluorescent droplet deposition experiment. Out of the 56 subjects tested, 42 (75%) passed filtering testing with the snorkel mask compared to 31 (55%) with a FFP3 respirator mask (p = 0.003). Amongst the 43 subjects who were not excluded following a placebo control, 85% passed filtering testing with the snorkel versus to 68% with a FFP3 mask (p = 0.008). Following front and lateral spray of fluorescence liquid particles, the snorkel mask also provided superior protection against droplet deposition within the subject’s face, when compared to a standard PPE combination of FFP3 masks and eye protection (3.19x108 versus 6.81x108 fluorescence units, phttps://www.ImperialHackspace.com/COVID-19-Snorkel-Respirator-Project/. Conclusion Full-face snorkel masks adapted as particulate respirators performed better than a standard PPE combination of FFP3 mask and eye protection against aerosol inhalation and droplet deposition. This adaptation is therefore a promising PPE solution for healthcare workers during highly contagious viral outbreaks.

Acute myocardial ischaemia and reperfusion (I–R) are major causes of ventricular arrhythmias in patients with a history of coronary artery disease. Ursodeoxycholic acid (UDCA) has previously been shown to be antiarrhythmic in fetal hearts. This study was performed to investigate if UDCA protects against ischaemia-induced and reperfusion-induced arrhythmias in the adult myocardium, and compares the effect of acute (perfusion only) versus prolonged (2 weeks pre-treatment plus perfusion) UDCA administration. Langendorff-perfused adult Sprague–Dawley rat hearts were subjected to acute regional ischaemia by ligation of the left anterior descending artery (10 min), followed by reperfusion (2 min), and arrhythmia incidence quantified. Prolonged UDCA administration reduced the incidence of acute ischaemia-induced arrhythmias (p = 0.028), with a reduction in number of ventricular ectopic beats during the ischaemic phase compared with acute treatment (10 ± 3 vs 58 ± 15, p = 0.036). No antiarrhythmic effect was observed in the acute UDCA administration group. Neither acute nor prolonged UDCA treatment altered the incidence of reperfusion arrhythmias. The antiarrhythmic effect of UDCA may be partially mediated by an increase in cardiac wavelength, due to the attenuation of conduction velocity slowing (p = 0.03), and the preservation of Connexin43 phosphorylation during acute ischaemia (p = 0.0027). The potential antiarrhythmic effects of prolonged UDCA administration merit further investigation.

We describe a human and large animal Langendorff experimental apparatus for live electrophysiological studies and measure the electrophysiological changes due to gap junction uncoupling in human and porcine hearts. The resultant ex vivo intact human and porcine model can bridge the translational gap between smaller simple laboratory models and clinical research. In particular, electrophysiological models would benefit from the greater myocardial mass of a large heart due to its effects on far-field signal, electrode contact issues and motion artefacts, consequently more closely mimicking the clinical setting. Porcine (n = 9) and human (n = 4) donor hearts were perfused on a custom-designed Langendorff apparatus. Epicardial electrograms were collected at 16 sites across the left atrium and left ventricle. A total of 1 mM of carbenoxolone was administered at 5 ml/min to induce cellular uncoupling, and then recordings were repeated at the same sites. Changes in electrogram characteristics were analysed. We demonstrate the viability of a controlled ex vivo model of intact porcine and human hearts for electrophysiology with pharmacological modulation. Carbenoxolone reduces cellular coupling and changes contact electrogram features. The time from stimulus artefact to (-dV/dt)max increased between baseline and carbenoxolone (47.9 ± 4.1–67.2 ± 2.7 ms) indicating conduction slowing. The features with the largest percentage change between baseline and carbenoxolone were fractionation + 185.3%, endpoint amplitude − 106.9%, S-endpoint gradient + 54.9%, S point − 39.4%, RS ratio + 38.6% and (-dV/dt)max − 20.9%. The physiological relevance of this methodological tool is that it provides a model to further investigate pharmacologically induced pro-arrhythmic substrates.

Background The underlying mechanism of ventricular fibrillation (VF) remains unclear. There are both experimental and clinical data to support the existence of rotational drivers (RDs), though other opposing studies suggest that VF is the result of disorganized myocardial activation. Abnormal electrical coupling between cardiomyocytes through gap junctions (GJ) has been considered an important factor in the genesis and maintenance of VF and pre-treatment with GJ couplers, rotigaptide (RTG), has been shown to reduce VF inducibility. Purpose We hypothesized that the degree of GJ coupling determines the underlying mechanism of VF, and that changes in GJ coupling can shift or modify the predominant mechanism of fibrillation along the spectrum between disorganised activity and organised drivers. We proposed that increased organisation of VF is critical to its termination. Methods Thirty Sprague-Dawley rat hearts were explanted, perfused ex-vivo and acute VF was induced with burst pacing and 30μM pinacidil. Optical mapping of transmembrane potential was performed at baseline and the effects of GJ coupling on VF dynamics were studied in an acute VF model by perfusing with increasing concentrations of a GJ uncoupler; carbenoxolone (0–50μM, CBX, n=10) or a GJ coupling-enhancer; RTG (0–80nM, n=10). A chronic diffuse fibrosis model (n=10) was generated with 4 weeks of in-vivo angiotensin infusion (500nm/kg/min). Fibrillation dynamics were quantified using phase analysis, phase singularity (PS) tracking and our novel method of global fibrillation organisation quantification, frequency dominance index (FDI), which is a power ratio of highest amplitude dominant frequency in the frequency spectrum. Results RTG increased average rotations per RD (Baseline: 2.86±0.10 vs 80nM: 5.66±0.43, p5 rotations. FDI increased with RTG (0.53±0.04 vs 0.78±0.3, p Conclusion The degree of GJ coupling is a key determinant of the underlying mechanism of VF. RTG organised fibrillation and stabilised RDs in a concentration-dependent manner whilst CBX disorganised VF. Enhancing GJ coupling with RTG in diseased hearts with fibrosis can terminate VF and may be a potential therapeutic target in acute VF. Acknowledgement/Funding BHF Programme Grant PG/16/17/32069

Background Ventricular fibrosis is known to play a critical role in initiation and maintenance of ventricular fibrillation (VF). Post myocardial infarction the quantity of fibrosis negatively correlates with survival. There is a lack of data on how the quantity and degree of fibrosis influences the mechanisms of VF itself. VF mechanisms remain debated, there are data to support both critical areas sustaining rotational drivers (RDs) and the contrary hypothesis of disorganized myocardial activation driving VF. Purpose We hypothesized that the underlying mechanism of VF is influenced by the spatial distribution and quantity of ventricular fibrosis. Methods Thirty-five Sprague-Dawley rats underwent permanent left anterior descending (LAD) ligation (n=11), 20mins LAD territory ischaemia-reperfusion (n=13) or in-vivo angiotensin infusion (500ng/kg/min, n=11) to generate compact (CF), patchy (PF) and diffuse fibrosis (DF) models respectively. After a 4-week maturation period, the hearts were explanted, Langendorff perfused and VF induced with burst pacing and 30μM pinacidil. Fibrillation dynamics were quantified using phase analysis, phase singularity (PS) tracking and our novel method of global fibrillation organisation quantification, frequency dominance index (FDI), which is a power ratio of highest amplitude dominant frequency in the frequency spectrum. Results Ventricular fibrosis for each group was characterized and quantified (CF: 22.3±3.2%, PF: 18.4±4.2%, DF: 5.8±1.3%, p=0.046). VF was driven predominantly by disorganised activity in CF, PSs were detected 26±7% of time comparative to 51.2±4% in DF and 69.5±8% in PF group (p=0.001). PF stabilised RDs, average maximum rotations for a single RD in PF were 31.6±7.1 comparative to 12.5±1.7 in DF and 6.4±1.1 in CF, p Conclusion VF mechanisms occur along a spectrum between organised activity sustained by discrete drivers and disorganised myocardial activation. The underlying VF mechanism can differ significantly dependent on the quantity and pattern of fibrosis. Patchy fibrosis stabilises RDs with localization to discrete areas and sustains an organised form of VF comparative to CF where VF is largely disorganised. Characterising the degree and pattern of fibrosis in patient groups vulnerable to VF might be beneficial in identifying patients with suitable targets for ablation. Acknowledgement/Funding BHF Programme Grant PG/16/17/32069

Background Mechanisms that maintain ventricular fibrillation (VF) remain debated. There are data supporting both critical areas sustaining rotational drivers (RDs) and the contrary hypothesis of disorganised myocardial activation driving VF. Abnormal electrical coupling between cardiomyocytes through gap junctions (GJ) and ventricular fibrosis are major factors in disease related remodelling, and have been implicated in initiation and maintenance of VF. In this study we hypothesised that the mechanism of VF is directly altered by fibrosis and GJ coupling, and that modulating these factors shifts or changes the predominant mechanism of fibrillation along the spectrum between disorganised activity and organised drivers. Methods VF optical mapping was performed in fifty-five perfused rat hearts with differing degrees and patterns of chronic ventricular fibrosis [compact (CF, n=11), patchy (PF, n=13) and diffuse (DF, n=11)], and acute GJ coupling modulation with rotigaptide (RTG, GJ coupling enhancer, 0–80nM, n=10) or carbenoxolone (CBX, GJ uncoupler, 0–50μM, n=10). Fibrillation dynamics were quantified using phase analysis, RD tracking and our novel method of global fibrillation organisation quantification, frequency dominance index (FDI), which is defined as the power ratio of highest amplitude dominant frequency in the frequency spectrum. Results In the fibrosis group, VF was driven predominantly by disorganised activity in CF, RDs were detected 26±7% of time comparative to 51.2±4% in DF and 69.5±8% in PF group (p=0.001). PF stabilised RDs, average maximum rotations for a single RD in PF were 31.6±7.1 comparative to 12.5±1.7 in DF and 6.4±1.1 in CF, p Conclusion VF mechanisms occur along a spectrum between organised activity with discrete drivers and disorganised myocardial activation. The degree of GJ coupling and ventricular fibrosis are key determinants of the underlying mechanism of VF. Enhanced GJ coupling and patchy fibrosis organised fibrillation and stabilised RDs, whilst GJ uncoupling and compact fibrosis disorganised VF. This study presents a unifying explanation for the numerous mechanisms reported for sustaining fibrillation. Characterising the degree and pattern of fibrosis in patient groups vulnerable to VF might be beneficial in identifying patients with targetable substrate, and GJ modulation might be a potential therapeutic target. Conflict of interest Nil

Summary Chimeric antigen receptor anti-CD19 (CAR19)-T cell immunotherapy-induced clinical remissions in CD19+ B cell lymphomas are often short lived. We tested whether CAR19-engineering of the CD1d-restricted invariant natural killer T (iNKT) cells would result in enhanced anti-lymphoma activity. CAR19-iNKT cells co-operatively activated by CD1d- and CAR19-CD19-dependent interactions are more effective than CAR19-T cells against CD1d-expressing lymphomas in vitro and in vivo. The swifter in vivo anti-lymphoma activity of CAR19-iNKT cells and their enhanced ability to eradicate brain lymphomas underpinned an improved tumor-free and overall survival. CD1D transcriptional de-repression by all-trans retinoic acid results in further enhanced cytotoxicity of CAR19-iNKT cells against CD19+ chronic lymphocytic leukemia cells. Thus, iNKT cells are a highly efficient platform for CAR-based immunotherapy of lymphomas and possibly other CD1d-expressing cancers., Graphical Abstract, Highlights • Bespoke protocol for CAR19-iNKT cell transduction and clinical scale expansion • Higher CAR19-iNKT than CAR19-T cell expandability and killing of CD19+CD1d+ targets • CAR19-iNKT cell reactivity potentiation by αGalCer and ATRA • Prolonged survival and brain lymphoma eradication of CAR19-iNKT cell-treated mice, Rotolo et al. show that anti-CD19 chimeric antigen receptor (CAR19)-engineered CD1d-restricted invariant NKT cells (iNKT) are more effective than CAR19-T cells against CD1d-expressing lymphomas, including those in the brain. De-repression of CD1d expression further enhances the anti-tumor efficacy of CAR19-iNKT.

We report that subunits of human nuclear proteasomes carry a previously unrecognised, constitutive posttranslational modification. Subunits with this modification are not visualised by SDS-PAGE, which is used in almost all denaturing protein gel electrophoresis. In contrast, CTAB-PAGE readily visualises such modified subunits. Thus, under most experimental conditions, with identical samples, SDS-PAGE yielded gel electrophoresis patterns for subunits of nuclear proteasomes which were misleading and strikingly different from those obtained with CTAB-PAGE. Initial analysis indicates a novel modification of a high negative charge with some similarity to polyADP-ribose, possibly explaining compatibility with (positively-charged) CTAB-PAGE but not (negatively-charged) SDS-PAGE and providing a mechanism for how nuclear proteasomes may interact with chromatin, DNA and other nuclear components.

Exercise thallium-201 myocardial scintigraphy was performed in 23 patients with hypertrophic cardiomyopathy. Eighteen patients presented with chest pain which was a persistent symptom in 11. Selective coronary arteriography was performed in 16 patients and showed normal coronary arteries in 15 and insignificant luminal irregularities in one patient. Eighteen patients had abnormal scintigrams. Three had an abnormal distribution of tracer entirely attributable to asymmetric septal hypertrophy, whereas 15 had discrete tracer uptake defects which could not be explained solely by myocardial hypertrophy. In this latter group of patients three scintigraphic patterns were identified: (1) in 10 patients defects were seen in scintigrams immediately after exercise but not in delayed images obtained four to six hours later. Eight of these patients had chest pain. (2) Four patients had uptake defects seen in both initial and delayed images. One patient had chest pain. (3) In three patients, one of whom had chest pain, tracer defects were seen only in delayed images and were not apparent in the initial scintigrams. Chest pain occurred in eight out of 10 patients with scintigraphic evidence of myocardial ischaemia but was present in only three out of 13 patients with non-ischaemic scintigrams. The value of exercise thallium-201 myocardial imaging as a diagnostic technique in hypertrophic cardiomyopathy appears limited. Scintigraphic evidence of regional myocardial ischaemia in the absence of significant coronary artery disease, however, contributes to an understanding of the mechanism of angina production in patients with hypertrophic cardiomyopathy.

A study was performed in 13 patients with idiopathic mitral valve prolapse and in 21 control subjects to assess the effect of the cold pressor test on systolic motion of the mitral valve. A significant increase in blood pressure occurred in 10 patients with mitral valve prolapse and in 19 controls after immersion of one hand in ice-cold water. M-mode echocardiographic recordings from the mitral valve were obtained in all patients before, during, and after the cold pressor test, together with simultaneous phonocardiograms in selected patients. Nine out of 10 patients with mitral valve prolapse and a hypertensive response to the cold stimulus showed a significant increase in the depth of mitral valve prolapse during the cold pressor test whereas in three patients with mitral valve prolapse and no hypertensive response the depth of mitral valve prolapse did not change during cold stimulation. Three patients with previously demonstrated mitral valve prolapse had equivocal resting echocardiograms but developed diagnostic evidence of mitral valve prolapse during the cold pressor test. No evidence of mitral valve prolapse was seen in any of the control subjects before, during or after the cold pressor test. In four patients with mitral valve prolapse and a hypertensive response to cold stimulation the systolic click was delayed by the cold pressor test, whereas the time of the systolic click remained constant in the three patients whose blood pressure did not increase. It is concluded that the cold pressor test provides a stimulus sufficient to delay the onset and increase the depth of mitral valve prolapse, thereby enhancing the diagnostic sensitivity of echocardiography in this condition.

Four patients with cardiac tumours were investigated by gated cardiac blood pool radionuclide imaging and echocardiography. Contrast angiocardiography was performed in three of the cases. Two left atrial tumours were detected by all three techniques. In one of these cases echocardiography alone showed additional mitral valve stenosis, but isotope imaging indicated tumour size more accurately. A large septal mass was detected by all three methods. In this patient echocardiography showed evidence of left ventricular outflow obstruction, confirmed at cardiac catheterisation, but gated isotope imaging provided a more detailed assessment of the abnormal cardiac anatomy. In the fourth case gated isotope imaging detected a large right ventricular tumour which had not been identified by echocardiography. Gated cardiac blood pool isotope imaging is a complementary technique to echocardiography for the non-invasive detection and assessment of cardiac tumours.