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A 57-year–old woman had previously undergone a right hemicolectomy for Duke B adenocarcinoma of caecum. She presented with acute back pain. A magnetic resonance imaging scan of her thoracic spine showed destruction of a vertebra with a tumor deposit at T11 (Figure 1A). She underwent a course of radiotherapy (20 Gray in 5 fractions) to this area. Good symptomatic relief was obtained. Biopsy demonstrated a sarcomatoid tumor suggestive of leiomyosarcoma. Over the next 2 months she developed dyspnea on exertion, fatigue, and leg edema. Examination revealed a regular tachycardia, raised jugular venous pressure, and bilateral leg edema. On auscultation, an added early diastolic sound (consistent with tumor plop) was audible. A 3-dimensional echocardiogram identified a large mass protruding from the right atrium into the right ventricle (Figure 1B and online-only Data Supplement Movie I). The mass prolapsed through the tricuspid valve into the right ventricle during diastole, intermittently obstructing right ventricular inflow and almost completely occluding the tricuspid valve …

Background— Takotsubo cardiomyopathy is an acute heart failure syndrome characterized by myocardial hypocontractility from the mid left ventricle to the apex. It is precipitated by extreme stress and can be triggered by intravenous catecholamine administration, particularly epinephrine. Despite its grave presentation, Takotsubo cardiomyopathy is rapidly reversible, with generally good prognosis. We hypothesized that this represents switching of epinephrine signaling through the pleiotropic β 2 -adrenergic receptor (β 2 AR) from canonical stimulatory G-protein–activated cardiostimulant to inhibitory G-protein–activated cardiodepressant pathways. Methods and Results— We describe an in vivo rat model in which a high intravenous epinephrine, but not norepinephrine, bolus produces the characteristic reversible apical depression of myocardial contraction coupled with basal hypercontractility. The effect is prevented via G i inactivation by pertussis toxin pretreatment. β 2 AR number and functional responses were greater in isolated apical cardiomyocytes than in basal cardiomyocytes, which confirmed the higher apical sensitivity and response to circulating epinephrine. In vitro studies demonstrated high-dose epinephrine can induce direct cardiomyocyte cardiodepression and cardioprotection in a β 2 AR-Gi–dependent manner. Preventing epinephrine-G i effects increased mortality in the Takotsubo model, whereas β-blockers that activate β 2 AR-G i exacerbated the epinephrine-dependent negative inotropic effects without further deaths. In contrast, levosimendan rescued the acute cardiac dysfunction without increased mortality. Conclusions— We suggest that biased agonism of epinephrine for β 2 AR-G s at low concentrations and for G i at high concentrations underpins the acute apical cardiodepression observed in Takotsubo cardiomyopathy, with an apical-basal gradient in β 2 ARs explaining the differential regional responses. We suggest this epinephrine-specific β 2 AR-G i signaling may have evolved as a cardioprotective strategy to limit catecholamine-induced myocardial toxicity during acute stress.

The human myocardium consists of a variety of different cell types. The cell that has been most extensively studied is the cardiomyocyte, which represents the single contracting unit of the myocardium. It has been estimated that there are 3 billion cardiomyocytes in the human ventricular myocardium, organized into the complex three-dimensional architecture of the ventricular myocardial tissue. Conceptually there are two underlying pathophysiological problems at the level of the cardiomyocyte which drive the functional deterioration of the failing heart. The first is numerical loss of cardiomyocytes, due both to the underlying causal disease process, such as acute myocardial infarction or chemotherapy, and to further loss secondary to apoptosis and necrosis triggered by theneurohormonal and inflammatory activation in the failing myocardium irrespective of the initial injury. Secondly, the remaining viable cardiomyocytes are required to provide sufficient contractile force to maintain an adequate cardiac output, despite loss of significant numbers and abnormal stress–strain relationships resulting from altered chamber geometry and extracellular matrix remodelling. The surviving cardiomyocytes compensate temporarily via transition to an adaptive hypertrophic state, which is associated with activation of fetal gene expression patterns. However, the persistent drive from the neurohormonal activation to maintain cardiac output, and activation of systemic inflammatory systems secondary to tissue hypoperfusion and congestion, combined with reduced metabolic efficiency in the hypertrophied cardiomyocytes, results in the development of contractile dysfunction of the hypertrophied cardiomyocyte. This is independent of the underlying cause of heart failure (HF), some of which cause further continuing impairment of cardiomyocyte function, such as familial dilated cardiomyopathy due to mutations in the cytoskeletal or sarcomeric proteins. The common endpoint for the failing heart, irrespective of underlying aetiology, is characterized at a cellular and molecular level by a distinctive failing signature. Cardiomyocytes isolated from failing human and animal hearts demonstrate impairment of both contraction (inotropy) and relaxation (lusitropy). Mechanical dysfunction of failing cardiomyocytes is amplified at higher beating frequencies, increased calcium concentration or during catecholamine stimulation. The loss of the force-frequency response (the Treppe or Bowditch effect) is a hallmark characteristic of HF. A variety of different pathophysiological factors have been proposed to underlie these mechanical alterations, including abnormal morphological and structural remodelling, and functional changes in signalling pathways, ionic fluxes, organelle function, and gene expression. It is becoming increasingly apparent that these are not independent processes; rather, crosstalk and interplay between the different pathophysiological processes serve to amplify the abnormalities and drive the deterioration of the failing cardiomyocyte phenotype.

Oblique Plane Microscopy (OPM) is a light sheet microscopy technique that combines oblique illumination with correction optics that tilt the focal plane of the collection system. OPM can be used to image conventionally mounted specimens on coverslips or tissue culture dishes and has low out-of-plane photobleaching and phototoxicity. No moving parts are required to achieve an optically sectioned image and so high speed optically sectioned imaging is possible. The first OPM results obtained using a high NA water immersion lens on a commercially available inverted microscope frame are presented, together with a measurement of the achievable optical resolution.

Cardiovascular diseases are complex pathologies that include alterations of various cell functions at the levels of intact tissue, single cells and subcellular signalling compartments. Conventional techniques to study these processes are extremely divergent and rely on a combination of individual methods, which usually provide spatially and temporally limited information on single parameters of interest. This review describes scanning ion conductance microscopy (SICM) as a novel versatile technique capable of simultaneously reporting various structural and functional parameters at nanometre resolution in living cardiovascular cells at the level of the whole tissue, single cells and at the subcellular level, to investigate the mechanisms of cardiovascular disease. SICM is a multimodal imaging technology that allows concurrent and dynamic analysis of membrane morphology and various functional parameters (cell volume, membrane potentials, cellular contraction, single ion-channel currents and some parameters of intracellular signalling) in intact living cardiovascular cells and tissues with nanometre resolution at different levels of organization (tissue, cellular and subcellular levels). Using this technique, we showed that at the tissue level, cell orientation in the inner and outer aortic arch distinguishes atheroprone and atheroprotected regions. At the cellular level, heart failure leads to a pronounced loss of T-tubules in cardiac myocytes accompanied by a reduction in Z-groove ratio. We also demonstrated the capability of SICM to measure the entire cell volume as an index of cellular hypertrophy. This method can be further combined with fluorescence to simultaneously measure cardiomyocyte contraction and intracellular calcium transients or to map subcellular localization of membrane receptors coupled to cyclic adenosine monophosphate production. The SICM pipette can be used for patch-clamp recordings of membrane potential and single channel currents. In conclusion, SICM provides a highly informative multimodal imaging platform for functional analysis of the mechanisms of cardiovascular diseases, which should facilitate identification of novel therapeutic strategies.

s S235 level of SDF-1a at explant. CXCR4 expression remained unchanged following CF-LVAD support, however it tended to be lower in DM pts suggesting an impaired ability in endogenous stem cell mobilization. Conclusion: Chronic LVAD therapy down regulates SDF-1a expression likely as a consequence of effective unloading. DM pts exhibit the lowest levels of SDF-1a. Low SDF-1a expression may lead to impaired mobilization of endogenous stem cells and negatively affect myocardial recovery. Whether therapeutic SDF-1 administration in this setting could promote recovery should be further investigated.

After chronic myocardial infarction (CMI) the myocardium undergoes remodeling which is described initially as ‘hypertrophic’ followed by ‘de-compensation’. We have studied this phenomenon at the sarcomeric level to understand how muscle mechanics, post-translational modifications, tissue morphology and sarcomeric structures are affected in the remaining well-perfused tissue.We use a rat model of left anterior descending (LAD) ligation to create phenotypic chronic myocardial infarction (CMI). A specialized temperature jump (T-jump) approach assess changes to myocardial muscle function during physiological shortening in saturating (32μM) and physiological (1μM) calcium concentrations. During ‘compensation’ we observe higher power (45%) compared to control. During ‘de-compensation’ we observe a 50% reduction in peak power and 40% reduction in maximal unloaded shortening velocity (Vmax). We exploit second harmonic generation (SHG) by two-photon excitation microscopy (TPE) to assess collagen abundance and infiltration. Global type I collagen abundance and infiltration increases 2-fold post-AMI but does not change during disease progression. Phos-Tag SDS-PAGE is used to assess Troponin I (TnI), Myosin Binding Protein C (MyBP-C) and Regulatory Light Chain (RLC) phosphorylation. Electron microscopy is used to assess sarcomeric organization and mitochondrial health and abundance. Sarcomeric disorder becomes evident with disease progression as well as a global reduction in functional mitochondria. This study demonstrates that sarcomeric protein modification is an important modifier of cardiac muscle function during the evolution of disease, which mirrors the route of progression from ‘compensated’ to ‘decompensated’ phenotypes that is part of the wider picture of heart failure that encompasses changes to tissue structure and organelle morphology.

Polarization of the mitochondrial membrane potential (DeltaPsi(m)) is critical for normal mitochondrial function and cellular energetics. Mitochondrial dysfunction, manifesting as disrupted DeltaPsi(m) polarization (i.e. depolarization or hyperpolarization), underlies several important and highly prevalent diseases, including a variety of cardiac and neurological disorders. As such, DeltaPsi(m) instability might form a unifying mechanism for a class of metabolic disorders affecting excitable tissues. Here, we measured the spatio-temporal kinetics of DeltaPsi(m) changes across the intact heart using high-resolution optical DeltaPsi(m) imaging and uncovered surprisingly complex spatial patterns and dynamically fluctuating changes in DeltaPsi(m) that developed into actively propagating waves of mitochondrial depolarization during global ischemia. Our data further indicated that the recovery of DeltaPsi(m) upon reperfusion is dictated by the duration of the preceding ischemic insult. Post-ischemic electrical and functional recovery was dependent on early DeltaPsi(m) recovery but independent of overall cellular injury measured using a standard assay of lactate dehydrogenase release. These findings reveal a novel mechanism by which instabilities in cellular energetic properties that are independent of irreversible cellular injury can scale to the level of the intact organ via an organized process of active conduction involving the multi-cellular network. This highlights the importance of investigating cellular metabolic properties in the context of the intact organ.

A 73-year-old Caucasian woman admitted to the neurology ward with Guillain–Barre syndrome (GBS) developed acute breathlessness 7 days after the onset of symptoms. Initial presentation was with symmetrical lower limb weakness, patchy sensory loss, absent tendon reflexes, normocellular cerebrospinal fluid (CSF) with raised protein (1.05 gm/l) and absent of waves on electromyography. There were no other associated cardiac symptoms, no neuromuscular respiratory weakness, normal neck muscle power, stable vital capacity and no hypercapnia on arterial blood gas analysis. She was a smoker. No arrhythmias were detected with continuous electrocardiogram (ECG) monitoring that is initiated on admission in the view of the well-recognized risk of autonomic dysfunction with GBS.1 On examination, she was apyrexial, well perfused, in sinus rhythm at a rate of about 70 beats/min and normotensive. There was congestive cardiac failure with elevated jugular venous pressure, bibasal crepitations and a fourth heart sound. Her ECG (Figure 1b) demonstrated downsloping ST segments, T-wave inversion in inferior and precordial leads and a prolonged corrected QT interval at 490 ms. A portable transthoracic echocardiogram revealed apical and antero-septal hypokinesia with impairment of left ventricular function. Troponin was mildly elevated (0.06 ng/ml, normal range

The peripheral type benzodiazepine receptor (mBZR) is a key mediator of mitochondrial dysfunction during oxidative stress. In isolated cardiomyocytes agonists of the mBZR cause ΔΨ m depolarization, KATP channel activation, and subsequent loss of cellular excitability. However, the spatiotemporal dynamics of ΔΨ m in the intact heart and their relationship to arrhythmias are unknown. Here, we investigate the mechanistic link between mBZR and ΔΨ m directly using adenoviral mediated gene transfer of the mBZR encoding gene (PKBS) in rat myocardium. Methods : Optical imaging was performed at a subcellular resolution in ex vivo perfused hearts from AdPKBS (n=5), AdGFP (n=2), and normal (n=4) rats. Normalized ΔΨ m was measured using a novel quantitative technique of TMRM imaging during global ischemia (7.5 min) followed by reperfusion. Mean and SD of baseline corrected & normalized ΔΨ m across 6400 sites were compared between groups. Results : In sharp contrast to normal and GFP groups, 4 of 5 PKBS hearts exhibited a paradoxical increase in ΔΨ m during ischemia (Fig A ) as ΔΨ m was significantly (pm observed in all groups during early ( ). Finally, ΔΨ m heterogeneity indexed by SD was markedly greater in PKBS compared to GFP and normal hearts during ischemia (p ). Conclusion : PKBS alters spatiotemporal dynamics of ΔΨ m and increases ΔΨ m heterogeneity across the intact heart. Modulating PKBS expression may be a novel therapeutic strategy for arrhythmias. Figure 1. Influence of PKBS gene transfer on myocardial ΔΨm during ischemia and reperfusion. Mean myocardial ΔΨm ( A ) and spatial Δψm heteroogeneity ( B ) during ischemia and reperfusion.

Effective clinical delivery of gene therapy to the heart requires understanding and design of complex biological systems to deliver therapeutic gene expression. The development of vectors that specifically target the myocardium, in particular bioengineered recombinant viruses, has improved the efficiency of gene delivery to the heart. These tools, coupled with advances in selection and design of the genetic payload, have led to effective cardiac gene therapy in preclinical models. This technology is currently translating to the clinic with a new wave of gene therapy trials for myocardial disease.

Stress cardiomyopathy, also referred to as Takotsubo cardiomyopathy, is an increasingly recognized clinical syndrome characterized by acute reversible apical ventricular dysfunction. We hypothesize that stress cardiomyopathy is a form of myocardial stunning, but with different cellular mechanisms to those seen during transient episodes of ischemia secondary to coronary stenoses. In this syndrome, we believe that high levels of circulating epinephrine trigger a switch in intracellular signal trafficking in ventricular cardiomyocytes, from G(s) protein to G(i) protein signaling via the beta(2)-adrenoceptor. Although this switch to beta(2)-adrenoceptor-G(i) protein signaling protects against the proapoptotic effects of intense activation of beta(1)-adrenoceptors, it is also negatively inotropic. This effect is greatest at the apical myocardium, in which the beta-adrenoceptor density is greatest. Our hypothesis has implications for the use of drugs or devices in the treatment of patients with stress cardiomyopathy.

Introduction The treatment of heart failure is entering a new era with the potential for gene therapy to directly target the underlying molecular abnormalities of the failing myocyte. One barrier to delivering gene therapy is the exclusion of patients who have neutralising antibodies (nAbs) to the recombinant viral vector due to previous exposure to the wild type virus. Aims To review the prevalence of nAbs against adeno-associated virus serotype 1 (AAV1) in published literature and to prospectively determine the prevalence of nAbs in a UK cohort of heart failure patients. Methods We reviewed the literature using the following search terms on Pubmed: (prevalence, neutralising antibodies, adeno-associated virus, AAV) which resulted in 19 studies. Five of these studies were suitable and provided data for 14 countries and a total of 1984 people. In addition, a total of 155 samples from heart failure patients at our institution were analysed for the presence of nAbs using a titre cut-off of 1:2. Results Globally there is a wide variation in the prevalence of nAbs to AAV1, ranging from 13 to 79%. These data are generally from young healthy volunteers and there is no standard cut-off for what titre constitutes a nAb positive result, though a titre cut-off of 1:20 was used in most of these studies. From our UK based heart failure cohort, 60.6% of patients were nAb positive at a titre of 1:2 and hence ineligible for gene therapy. The only characteristic associated with being nAb positive was age with the prevalence of nAbs increasing significantly with age (Table 1). Conclusion If gene therapy proves to be an effective treatment, then currently three-fifths of heart failure patients in the UK may be ineligible to receive it due to presence of nAbs. This barrier seems to be greater in the older patients. Published data on the prevalence of nAbs does, however, suggest there is a wide geographic variation. Strategies are in development to overcome the barrier of nAbs but until these are effective we must be conscious of this limitation when discussing gene therapy as either a research or therapeutic option with our patients in the UK.

We are developing an autofluorescence (AFL) lifetime-based technique to characterise the signatures associated with histological, metabolic and functional changes in myocardial disease. AFL spectroscopy exploits the properties of a number of endogenous molecules and offers the potential to avoid exogenous label use that could potentially alter the physiological environment. AFL measurements of molecules such as NADH and flavins may report energetic state changes, whilst signatures from matrix such as collagen inform of structural alterations. We investigated the application of a fibre-optic based single-point instrument combining time-resolved spectrofluorometry and diffuse reflectance spectroscopy to a doxorubicin cardiomyopathy heart failure model (DOX-HF) in vivo . Sprague-Dawley male rats received 1.25 mg/kg doxorubicin or vehicle (0.9% NaCl) via tail vein injection weekly for 8 weeks. AFL signals were measured in vivo in week 11, once heart failure phenotype was well established. DOX-HF(n = 8) LVEF 49.6% vs. 80.4% in controls (n = 4)(**p = 0.006). At 11 weeks, significant differences in AFL signals between DOX-HF(n = 6) and control (n = 3) in LV, RV and LV posterior wall were observed (*p Our instrument has sensitivity to characterise in vivo changes in DOX-HF model without use of exogenous compounds. Incorporation into coronary catheters or pacing leads could offer additional diagnostic information and monitoring. Identification of earlier changes could permit development of a screening strategy to identify cancer patients developing cardiomyopathy to permit judicious adjustment of chemotherapy.

e21516 Background: Doxorubicin (Dox.) (alone or in combination) is the standard first line therapy for metastatic soft tissue sarcoma (STS). Cardiotoxocity is an important complication of dox., and is often defined as left ventricular ejection fraction (LVEF) 10%. Studies in patients treated with high dose chemotherapy have suggested that elevated troponin levels predict the development of cardiotoxicity. However, conflicting data exists for conventional dose chemotherapy. We aimed to evaluate the utility of troponin to predict cardiotoxicity in metastatic STS patients treated with dox. Methods: Over 18 months, all patients with metastatic STS receiving single agent dox. received echocardiograms and serum troponin I (TnI) levels at baseline and following cycles 2, 4 and 6. TnI was measured using the Beckmann Coulter Access 2 assay with a normal range of < 40 ng/L. Results: 41 patients (28 female, 13 males), median age 58 years, were studied. 11 (27%) had cardiac risk factors. Median num...

Background Heart failure with preserved ejection fraction (HF-PEF) is common and has a poor prognosis with a 3 year mortality rate of 23%. There are currently no effective therapies for this condition. HF-PEF is characterized by symptoms of heart failure, normal or a mildly impaired ejection fraction and evidence of adverse cardiac and vascular remodelling. A feature of all heart failure is a heightened sympathetic nervous system (SNS), which can now be abrogated using renal denervation (RD). Methods We are conducting a study of patients with HF-PEF who will be randomised (2:1) to receive RD or open control. As a phase II mechanistic study we are primarily investigating the effect of RD on patient symptoms (questionnaire), cardio-pulmonary exercise function, B-type natriuretic peptide levels, left ventricular filling pressures, left ventricular mass and left atrial volume. We are also assessing macrovascular function using aorta imaging to calculate aorta distensibility, pulse wave velocity and aortic flow. Finally

We investigate the potential of an instrument combining time-resolved spectrofluorometry and diffuse reflectance spectroscopy to measure structural and metabolic changes in cardiac tissue in vivo in a 16 week post-myocardial infarction heart failure model in rats. In the scar region, we observed changes in the fluorescence signal that can be explained by increased collagen content, which is in good agreement with histology. In areas remote from the scar tissue, we measured changes in the fluorescence signal (p < 0.001) that cannot be explained by differences in collagen content and we attribute this to altered metabolism within the myocardium. A linear discriminant analysis algorithm was applied to the measurements to predict the tissue disease state. When we combine all measurements, our results reveal high diagnostic accuracy in the infarcted area (100%) and border zone (94.44%) as well as in remote regions from the scar (> 77%). Overall, our results demonstrate the potential of our instrument to characterize structural and metabolic changes in a failing heart in vivo without using exogenous labels.

Cardiac failure is characterised by the loss of cardiomyocytes, and several strategies to replace the lost cell mass are being developed. Animal models have demonstrated the therapeutic potential of several cell types, and both autologous skeletal myoblasts and bone marrow progenitor cells have been tested in preliminary clinical trials. However functional improvements have been modest and the mechanism of benefit is unclear, although myocardial regeneration is not a significant factor. Alternative strategies using autologous resident cardiac progenitor cells or embryonic stem cell-derived cardiomyocytes could recreate de novo myocardium with higher efficiency, although various hurdles must be overcome before these strategies are translated to the clinic.

We thank Steele et al . for their interest and comments regarding our recent study focusing on the effects of INa reduction on spontaneous sarcoplasmic reticulum (SR) Ca2+ release.1 In the context of the authors' previous publications regarding the mechanism of action of flecainide at the cardiac ryanodine receptor (RyR2),2–4 it appears they have interpreted our study as an attempt to infer that flecainide has no effect on RyR2. We would like to clarify this misinterpretation. Our work demonstrates that a reduction in Na+ influx into the cardiomyocyte can, via the enhancement of Ca2+ efflux through the Na+/Ca2+ exchanger, reduce [Ca2+]i in the vicinity of the RyR2 and thus reduce the frequency of spontaneous SR Ca2+ release events. We demonstrate that this is a class effect of INa blockers, and that flecainide causes no reduction in SR Ca2+ leak when INa is eliminated by altering the holding potential via voltage clamp. This mechanism also appears to be relevant in whole-heart models of arrhythmia induced by increased SR Ca2+ release as evidenced by recent data from Radwanski et al .5 Our data should not be taken as a dismissal of the effects of flecainide at RyR2. We merely conclude that, under our experimental conditions, INa blockade confers a greater reduction in spontaneous SR Ca2+ release than effects at the RyR2. Flecainide has been …

Time-resolved autofluorescence spectroscopy permits characterization of biological tissues and has already shown the potential in a number of disease applications. It harnesses the photo-physical properties of a number of endogenous molecules, e.g. NADH and flavoproteins, to offer an opportunity to discern the energetic state of the myocardium without the issues of toxicity or consequences associated with the introduction of exogenous compounds. Additionally, autofluorescence from extra-cellular matrix molecules, e.g. collagen, can provide information on structural changes to the heart. We report the development and application of a custom fibre-optic probe-based time-resolved spectrofluorometer combining time-correlated single photon counting detection and white-light reflectometry to characterize the autofluorescence ‘fingerprint’ associated with the changes in histology, morphology, metabolism and function in heart health and pathological states. Measurement of fluorescence lifetime is inherently ratiometric and unaffected by molecule concentration and excitation intensity amongst other experimental variables. Furthermore fluorescence lifetime is affected by changes in the microenvironment e.g. protein binding states. Explanted hearts from male SD rats perfused using the ex vivo Langendorff system demonstrated stable fluorescence lifetimes. Fluorescence lifetime signals were successfully derived from fluorescence decays with acquisition times as low as 1 second. The changes in the autofluorescence signal of ex vivo Langendorff preparations using perfusate column switching were characterised for an ischaemia-reperfusion model and for a change of substrate between glucose and mannitol. This technology has the potential to be a label-free method to observe biochemical changes associated with disease mechanisms in the heart, with clear potential application to cardiac research and clinical diagnostics.

Introduction: A major concern for patients receiving an implantable cardioverter defibrillator (ICD) is the rate of ICD shocks. This is an important consideration when counselling a patient prior to implantation but it is challenging to provide an accurate estimate due to the reported heterogeneity of this outcome in clinical trials and the common disjoint between clinical trials and real-world experience. An additional concern for physicians since the publication of MADIT-RIT is the rate of ATP therapy. Contemporary, real-world UK data in heart failure patients is lacking. Aim: We sought to determine the incidence of appropriate ICD therapy in our real world cohort of heart failure patients. Methods: Patients under active ICD follow-up in 2011 and 2012 were identified at two UK tertiary cardiology centres. Patients who had heart failure with an ejection fraction (EF) ≤35% (or severely impaired) were included. Each patient had at least 12 months follow-up data. Results: Data were analysed for 479 patients in 2011 and 537 patients in 2012: median age at implant 67years (IQR 58-73), median EF 25% (IQR 20-30). Underlying aetiologies were ischaemic (74%), DCM (15%) and unknown(11%). Indications were primary prevention (55%), secondary prevention (43%) or not documented (2%). Results are summarised in the table. Conclusions: There was no significant difference in the rates of ICD therapy (shock or ATP) between those patients with a primary or secondary prevention indication. On average the annual rate of appropriate shocks for primary and secondary prevention were 7.7% and 11.5% respectively. Contemporary data detailing rates of appropriate therapy in secondary prevention patients is particularly lacking and our data shows rate of appropriate shocks in the secondary prevention group was much lower than the some older trials (45% in the first year of the AVID trial). This highlights the importance of establishing contemporary event rates in order to best inform patients. Our data would suggest that the rate of ATP in our primary prevention cohort is comparable to that seen in the conventional arm of MADIT-RIT. As such it will be of interest to repeat this data collection now that more conservative programming has been implemented. | | Primary Prevention | Secondary Prevention | Fishers exact test (P=) | | ---------------------- | ------------------ | -------------------- | ----------------------- | | Appropriate shock 2011 | 8.2% | 14.1% | 0.052 | | Appropriate ATP 2011 | 14.3% | 15.7% | 0.743 | | Appropriate shock 2012 | 7.1% | 8.9% | 0.413 | | Appropriate ATP 2012 | 12% | 14.4% | 0.415 | Table showing the proportion of patients receiving ICD therapy annually in 2011/12 categorised by indication for ICD implantation.

Introduction: The autonomic nervous system (ANS) responds within seconds to homeostatic perturbations. Heart rate variability (HRV), though a widely accepted ANS measurement, fails to detect sudden changes as sampling windows of 5 minutes or more are typically used. Aim: We sought to determine whether 1 minute sampling windows for frequency-domain and non-linear HRV parameters could be used to describe rapid changes in the ANS accurately during tilt table testing (TTT). Methods: Frequency-domain parameters (LFnu, HFnu) and non-linear short term measures of variability (SD1 from the Poincare plot and α1 slope from detrended fluctuation analysis) were derived from patients undergoing clinically indicated TTTs. To determine reproducibility of 1 minute windows, a 5 minute rest period was analysed then divided into 1 minute intervals. Using 1minute windows we analysed the TTTs: 1minute before and 5 minutes after tilt and GTN then 5 minutes pre-syncope. In those without syncope an equivalent pre-syncope 5 minute time frame was estimated. Results: TTT assessments in 295 consecutive patients were reviewed. Subjects were excluded if they had unusable traces (e.g. atrial fibrillation, paced rhythms) leaving 84 patients without syncope (tilt negatives) and 27 tilt-induced syncope patients (tilt positives). All four HRV parameters were reproducible at rest using 1 minute windows. LFnu and α1 increased significantly while HFnu and SD1 fell during the 5 minutes after tilt-up and GTN administration. The changes post tilt-up were first detectable at 1-2 minutes (LFnu, HFnu) but there were no differences in any parameter with respect to tilt outcome negative/positive. However, in the pre-syncope period all parameters were significantly different between negative and positive tilts. The earliest difference recognised was at 3 minutes pre-syncope when, compared with tilt negatives, syncope patients displayed reduced LFnu (65.2±20.9 vs 77.3±19.0, p=0.04), increased HFnu (34.5±33.8 vs 22.6±18.8, p=0.04) and a reduced α1 (1.0±0.4 vs 1.4±0.3, p=0.004). Conclusions: One minute time windows may be used to analyse HRV with improved temporal sensitivity. Using this technique, we demonstrated changes consistent with sympathetic activation in patients at tilt-up and nitrate administration followed by a shift to vagal predominance pre-syncope in positive tests. Our work uniquely identifies changes in the frequency-domain parameters to occur early, between minute 1 and 2 of tilt-up, and that vagal activation could be detected as early as 3 minutes before syncope. This technique may be useful for detecting acute changes in the ANS in a variety of situations.

Purpose: Changes to the cellular expression of numerous microRNAs (miRs) have been implicated in disease development. MiR-1 expression is down-regulated in human heart failure (HF) and our in vivo rat HF model, we aimed to further understand its significance in HF. We previously showed that miR-1 expression was restored by in vitro adenovirus-mediated restoration of SERCA2a in adult rat HF cardiomyocytes, in response to diastolic calcium changes. Methods: Adult rats that underwent surgical ligation of the left anterior descending coronary artery to induce myocardial infarction (>30%) were maintained 16-20 weeks until HF developed. Cardiomyocytes were isolated for in vitro transfection by lipofection with miR-1 constructs followed by 48 hours culture. We examined cardiomyocyte calcium handling, contractile function, and gene expression analysis by quantitative RT-PCR. Results: The contractile beta-adrenoceptor (βAR) response to isoprenaline was depressed in cardiomyocytes from HF rats, but was restored in HF cardiomyocytes by increasing miR-1 expression (Figure 1) with a mature miR-1 construct. Selective pharmacological inhibition revealed that SERCA2a function was unchanged, and that it was an elevated β2AR response that had improved cell shortening in the HF cardiomyocytes to levels comparable to healthy. β2AR expression was unchanged after pre miR-1 treatment, implying it is the functional signalling of the receptor that is influenced by miR-1. Conclusion: Restoration of miR-1 reverses HF-induced βAR desensitisation. We suggest that the SERCA2a down-regulation and consequent increase of diastolic calcium may signal through miR-1 reduction to produce βAR signalling alterations in HF. ![Figure][1] miR-1 Restores Beta-AR Response in HF [1]: pending:yes

Rationale: The use of anthracyclines such as doxorubicin (DOX) has improved mortality and morbidity in cancer patients, yet associated risks of cardiomyopathy have limited their clinical application. DOX-associated cardiotoxicity is frequently irreversible and typically progresses to heart failure (HF). However, our understanding of molecular mechanisms essential for development of cardioprotective strategies and to predict HF remain largely obscure. Objective: As microRNAs (miRNAs) have been shown to play potent regulatory roles in both cardiovascular disease and cancer, we investigated miRNA changes in chemotherapy-induced HF and the alteration of cellular processes downstream. Methods and results: Myocardial miRNA profiling was performed after DOX-induced injury, either via acute application to isolated cardiomyocytes in vitro or chronic exposure in vivo, and also compared with miRNA profiles from remodeled hearts following myocardial infarction; the miR-30 family was down-regulated in all three models. We confirmed β1- and β2-adrenoceptors (β1AR, β2AR), Gi alpha 2 (Giα-2) and the pro-apoptotic gene BNIP3L/NIX as miR-30 targets. We describe regulation of the β-adrenergic pathway by miR-30, where a preferential βAR inhibition results in a β-blocker like effect. We demonstrate that high miR-30 levels are protective against DOX insult and correlate with lower reactive oxygen species (ROS) generation. Moreover, we identify GATA-6 as a mediator of the DOX-associated reduction in miR-30 expression. Conclusion: DOX causes acute and sustained miR-30 down-regulation in cardiomyocytes via GATA-6. miR-30 overexpression protected cardiac cells from DOX-induced apoptosis, and its maintenance represents a potential cardioprotective strategy for anthracycline cardiomyopathy. Additionally, intriguing synergic anti-cancer effects were observed for miR-30.

Background: Takotsubo cardiomyopathy (TCM) is an acute heart failure syndrome that can occur after a period of intense emotional stress, characterised by a hypokinetic apex and a hyperkinetic base. We have previously shown that a single IV injection of epinephrine can recapitulate the syndrome in male rats. As TCM usually occurs in post-menopausal females we gave the same epinephrine dose to normal female and ovariectomised (OVX) rats with or without short or long-term (2 weeks) estrogen replacement. Methods: OVX rats were anaesthetised with 2% isoflurane in 100% O2 and a parasternal long-axis echocardiograph was acquired. For short-term estrogen treatment, 600ng 17β-estradiol was infused IV 5 minutes before epinephrine injection (4.3x10-8mol.100g-1). For 2-week replacement, osmotic minipumps were implanted containing 17β-estradiol (238μg.day-1) in 50:50 glycerol:DMSO. Fractional shortening was measured before and at 5 minute intervals after epinephrine using M-mode images from the apex and base of the left ventricle. QTc was calculated using the LabChart 7 ECG Analysis Module from a Lead II ECG. Results: Compared to males, female non-OVX rats had reduced mortality and did not exhibit TCM-like contraction patterns when given the same epinephrine bolus (6.25% vs. 46% mortality; P

Introduction To determine the impact of internationally recognised criteria for the diagnosis of heart failure with preserved ejection fraction (HF-PEF) on the identification of patients suitable for a clinical study of a novel therapeutic intervention. Methods We reviewed the notes of 5883 patients admitted with heart failure across 8 hospitals in the UK. The criteria for diagnosis for HF-PEF included (Figure 1): ejection fraction (EF) >50%, structural remodelling, evidence of impaired diastolic function and the absence of significant other cause of symptoms, as recommended in the most recent ESC guidelines. Results Overall, 20.5% of patients had an EF >50% (Figure 2). There was significant heterogeneity between the hospitals as this proportion varied from 42.4% in the teaching hospitals to 13.8% in the district general hospitals (p Conclusion There may be a degree of under-diagnosis of HF-PEF in less specialised hospitals in the UK. Applying the current ESC criteria for HF-PEF for trial recruitment would limit enrolment to 3.7% of those who have an EF >50% and only 0.8% of all heart failure admissions. These data have important implications for the feasibility of clinical trials for HF-PEF, and for the generalisability of the results of such studies.

Purpose There are few non-invasive techniques to predict and monitor patients’ responses to left ventricular assist device (LVAD) therapy. MicroRNAs (miRs) are small noncoding RNAs with intricate roles in cardiovascular disease. They remain stable in the circulation, are readily quantified, and may be useful as new biomarkers. This study sought to identify candidate miR biomarkers for further investigation and to investigate whether these circulating miRs were of myocardial origin. Methods and Results We studied 55 serial plasma and myocardial samples from 19 patients who underwent HeartMate II LVAD implantation, and used a screening microarray to analyse the change in expression of 1,113 miRs from pre-implant and 6 months follow-up. Twelve miRs showed significant variation and underwent validation, with miR-1202 and miR-483–3p selected for further study. The key findings in the test cohort (n = 8) were: (1) miR-483–3p showed upregulation after 6 months of LVAD therapy in circulating plasma (median fold change 2.32 (1.30–2.44); p = 0.021) and in ventricular myocardium (median fold change 1.799 (0.717–4.719; p = 0.169). This mirrored the reduction in NT-proBNP levels at 6 months (fold change 0.30 (0.08–0.43); p = 0.004; see Figure). (2) Using change in NT-proBNP at 3 months as a marker of clinical response to LVAD therapy, baseline expression of plasma miR-1202 identified good versus poor LVAD responders (absolute expression 1.296 (1.293–1.306) vs. 1.311 (1.310–1.318) A.U.; p = 0.004). (3) Both miR-483–3p and miR-1202 are also enriched in ventricular myocardium suggesting the heart as the possible source of these plasma miRs. Conclusions This is the first report of circulating miR biomarkers in LVAD patients. We demonstrate the feasibility of this approach, and report the potential for miR-483–3p and miR-1202 respectively to monitor and predict response to LVAD therapy. We suggest that changes in miR-483–3p expression could provide a more specific assessment of ventricular function that complements the changes in systemic neuroendocrine milieu recorded by serial measurement of natriuretic peptides, and suggest that miR-1202 could be valuable for judging the likelihood of good response to LVAD support. However, these conclusions are weakened by small sample size and retrospective analysis. We propose further work to study these hypotheses further and elucidate roles for miR-483–3p and miR-1202 in clinical practice and in underlying biological processes.

Fluorescence spectroscopy is a promising tool for the characterisation of biological tissues and shows the potential for in vivo clinical diagnosis in many diseases. It exploits the inherent photo-physical properties of a number of endogenous molecules. Fluorescence lifetime measurements of these fluorophores such as NADH and flavoproteins present an opportunity to discern functional information concerning myocardial energetics without issues of toxicity or systemic effects associated with the introduction of exogenous compounds. Additionally, autofluorescence from extra-cellular matrix molecules e.g. collagen may provide information on structural changes to the heart. Measurements of fluorescence lifetime are independent of fluorophore concentration, excitation intensity, sample attenuation and other experimental artefacts and can also report on changes to the fluorophore microenvironment e.g. pH and protein binding state. Thus we are interested to develop autofluorescence lifetime (AFL)-based techniques as a myocardial "optical biopsy". We report the application of a custom fibre-optic probe-based time-resolved spectrofluorometer utilising time-correlated single photon counting that we developed to characterise the autofluorescence signatures associated with the histological, morphological, metabolic and functional changes in myocardial tissue in health and disease states. We studied an in vivo rat left anterior descending coronary artery ligation heart failure (MI-HF) model 16 weeks post-infarction, which is a model well characterised in our institution. We observed stable AFL signals across age-matched control (AMC) hearts (n = 6) in different anatomical locations. We observed significant differences in AFL signals between MI-HF (n = 6) and AMC (n = 6) in infarcted regions: left ventricle (LV) anterior wall (p in vivo application of time-resolved fluorescence spectroscopy to the study of the heart. This technique shows promise as a diagnostic tool and could be readily and rapidly translatable into clinical cardiology practice.

Nat. Rev. Cardiol. 10, 584–598 (2013); doi:10.1038/nrcardio.2013.114 In the version of this article originally published, the address for Alexander R. Lyon was incorrectly stated. The address should have been: NIHR Cardiovascular Biomedical Research Unit, Royal Brompton Hospital, Sydney Street, London, SW3 6NP, UK.

Background Gap junctional uncoupling is thought to contribute to arrhythmogenesis through its effects on conduction slowing and creation of heterogeneities, often in the context of action potential or structural abnormalities. However, it is uncertain as to whether moderate conduction slowing through gap junctional uncoupling on its own, in the absence of action potential or structural changes, is sufficient to be pro-arrhythmic. We used optical mapping: (1) to study the effects of carbenoxolone, a gap junction uncoupler, on conduction velocity and action potential duration, and (2) to assess the effects of moderate conduction slowing and gap junctional uncoupling on arrhythmia vulnerability in structurally normal hearts. Methods Normal rat hearts (n=12) were isolated and perfused with Krebs-Henseleit solution, then loaded with a voltage-sensitive dye (RH237) and perfused with an excitation-contraction uncoupler (10 μM blebbistatin) to eliminate motion artefact. Transmembrane voltage (V m ) transients were recorded using a 256-photodiode array and epicardial activation was mapped across the anterolateral left ventricle during ventricular pacing (stimulus 1 mA, cycle length 180 ms). Hearts were then perfused with 30 μM carbenoxolone (CBX; n=6) or control (n=6) for 30 min and epicardial activation mapped every 2 min. Subsequently, hearts were subjected to programmed ventricular stimulation with up to three extrastimuli and assigned an Inducibility Quotient depending on ease of arrhythmia inducibility. Results Carbenoxolone significantly slowed conduction across the left ventricular epicardial surface (Conduction velocity index: Baseline 100%, CBX 70% ± 2%, p Conclusions Gap junctional uncoupling with carbenoxolone produced a 30% reduction in conduction velocity without changes in action potential duration, and resulted in increased arrhythmia vulnerability in structurally normal hearts. These results suggest that moderate conduction slowing produced by gap junctional uncoupling in isolation, that is, in the absence of changes in action potential duration or structural abnormalities, is sufficient to increase susceptibility to ventricular arrhythmias, and therefore highlight the importance of gap junctional uncoupling in ventricular arrhythmogenesis.

British Journal of Hospital Medicine, July 2013, Vol 74, No 7 415 © 2 01 3 M A H ea lth ca re L td found. However, currently this remains an extremely rare occurrence, and at this time we do not think the diagnostic criteria, including reversibility, should be amended. This question also underlines the need for complete national and international registries of takotsubo cardiomyopathy that will help to provide more information to help answer these exact questions. Andrew C Morley-Smith Clinical Research Fellow National Heart and Lung Institute Imperial College and National Institute for Health Research Cardiovascular Biomedical Research Unit Royal Brompton Hospital London SW3 6NP Elmir Omerovic Associate Professor of Cardiology Department of Molecular and Clinical Medicine Sahlgrenska Academy at University of Gothenburg Division of Cardiology Sahlgrenska University Hospital Gothenburg Sweden Alexander R Lyon Senior Lecturer and Consultant Cardiologist National Heart and Lung Institute Imperial College and National Institute for Health Research Cardiovascular Biomedical Research Unit Royal Brompton Hospital London

Aim Ca2+ waves are thought to be important in the aetiology of ventricular tachyarrhythmias. There is some evidence that INa blocking agents can reduce spontaneous sarcoplasmic reticulum (SR) Ca2+ release via effects on the RyR. We tested the hypothesis that direct modulation of INa may also be important in altering SR Ca2+ release. Methods and Results Imaging of spontaneous SR Ca2+ release events in healthy adult rat cardiomyocytes was performed. Variation in frequency of stimulation was used to produce Ca2+ sparks or waves. When SR Ca2+ content was held constant, spark frequency, wave frequency and wave velocity were reduced by a variety of INa blockers (including flecainide, lidocaine, propafenone and TTX). To assess the contribution of INa to spark and wave production voltage clamping was used to activate contraction from holding potentials of -80mV or -40mV. This confirmed that reducing Na+ influx during myocyte stimulation is sufficient to reduce waves and that such agents only cause Ca2+ wave reduction when INa is active. It was found that Na+/Ca2+-exchanger (NCX)-mediated Ca2+ efflux was significantly enhanced by INa blockade and that the effects of INa blockade on wave frequency could be reversed by reducing [Na+]o, suggesting an important downstream role for NCX function in the changes in SR Ca2+ release. Veratridine, an INa activator, increased wave frequency and the effects were abrogated by increasing [Na]o during the wave-detection period. Conclusions Our results show, for the first time, that reducing Na+ influx reduces spontaneous SR Ca2+ release. We have also shown that alterations in [Na+]i modulate wave frequency through alterations in NCX function. A reduction in INa, for example, can increase the [Na+]o:[Na+]i gradient, hence causing more effective Ca2+ efflux at resting membrane potentials. This not only suggests a clear mechanism for the reduction in waves seen with INa blockers, but also emphasises the detrimental effects of high [Na]i seen in disease states such as heart failure. It also allows us to understand an alternative way in which INa blockers exert their antiarrhythmic actions.

Introduction Irreversible alveolar capillary membrane (ACM) remodelling accompanies chronic heart failure (CHF), contributing to dyspnoea, the predominant symptom that limits quality of life in CHF. Gene therapy is aimed at improving myocardial function in CHF. Restoration of Sarco-endoplasmic reticulum calcium ATPase (SERCA2a) gene expression in animal models of CHF restores haemodynamic parameters towards normal. The lungs are the direct upstream target of raised left atrial pressure and hence pulmonary venous hypertension. We hypothesised that mechanical strain at the pulmonary micro vasculature associated with PVH up-regulates mediators leading to pulmonary inflammation and ACM remodelling. We have previously shown that gene expression of monocyte chemoattractant protein (MCP)-1, interleukin(IL)-6, endothelin (ET)-1, endothelin receptors (ETR) A and B, and endothelial converting enzyme (ECE) are altered in the lungs of Sprague-Dawley rats at 16 weeks after left coronary artery ligation. We now sought to determine the effect of SERCA2a gene therapy on gene expression of these mediators in the lung. Methods Gene expression of components of the ET-1 pathway, MCP-1 and IL-6 were investigated in whole lungs of rats at 16 weeks after LCA, at 16 weeks post LCA with tail vein injection of adeno-associated viral (AAV) gene transfer of SERCA2a at 12 weeks post LCA, or sham procedure(n=5 in each group). Lungs were snap frozen in liquid nitrogen, RNA extracted using a modified Trizol and RN easy protocol and gene expression determined in reverse transcribed cDNA by qPCR. Results Expression of ET-1, ETAR, MCP-1 and IL-6 genes were elevated in heart failure animals and reduced to or towards normal in SERCA2a treated animals. In heart failure animals there was a trend towards reduced ETRB expression which was significantly improved by SERCA2a gene therapy (figure 1). ECE gene expression was not altered by LCA or gene therapy. Conclusion SERCA2a gene therapy directed at the myocardium in heart failure also affects gene expression in the lungs of CHF animals. This may provide therapeutic benefit to the lungs in addition, reducing inflammation and stimuli associated with structural and vascular remodelling.

Gap junction (GJ) coupling plays an important role in determining the electrophysiological properties of intact myocardium. Pharmacologically increasing coupling reduces the incidence of spontaneous and programmed arrhythmias and may have an anti-arrhythmic effect during acute ischaemia. A pro-arrhythmic model of regional ischaemia was developed in isolated perfused rat hearts (n=6/group (0, 15, 30, 60 min of LAD occlusion)). During ischaemia, all hearts experienced VPB, 60% experienced VT with arrhythmia incidence peaking at 12 min. On reperfusion, hearts undergoing 15 min of ischaemia were significantly pro-arrhythmic (Incidence of VT/VF 83% vs 60 min, 0%, p The 15-min model was used to assess the effects of increasing coupling, with Rotigaptide, on arrhythmia incidence during ischaemia and reperfusion. Hearts (n=9/group) were perfused with either (1) No drug (control), (2) Rotigaptide (50 nM) pre-occlusion, (3) Rotigaptide at occlusion or (4) Rotigaptide at reperfusion. Pre-treatment with Rotigaptide afforded a significant reduction in arrhythmia score during ischaemia (0.90±0.24 vs control 2.52±0.49, p In acute ischaemia, increasing GJ coupling, prior to, or, at the time of LAD occlusion reduced the burden of ventricular arrhythmias during ischaemia but did not alter reperfusion arrhythmogenesis. Increasing coupling in the early stage of regional ischaemia afforded the most substantial reduction in ventricular arrhythmias.

Background Clinical audit provides a mechanism to review the quality of patient care, and highlight areas for further development. The National Institute of Clinical Excellence (NICE) updated the clinical guidelines for chronic heart failure (HF) in August 2010. Incorporated into this update were audit criteria, which were used to create an electronic tool to assess current outpatient management. Methods The audit targeted patients with HF secondary to left ventricular disease attending a tertiary cardiac centre in London. After an initial pilot period using hard copy proformas, the audit was extended in electronic format to all HF clinics within the institution. This enabled the capture of demographical data, diagnosis, treatment, monitoring including heart rhythm and rate, measures of clinical follow-up, and the compliance with rehabilitation. Results A total of 282 patients were included in the 8-month audit, of which 71% were male. The majority of patients were elderly, with a mean age of 68 years, and most lived outside of London (68%). The three commonest HF aetiologies were ischaemic heart disease (40%), idiopathic dilated cardiomyopathy (20%) and primary valvular disease (12%). Treatment demonstrated 89% correctly prescribed a β-blocker, 91% correctly prescribed an ACE-inhibitor or angiotensin receptor antagonist and 64% correctly prescribed an aldosterone antagonist. Rehabilitation questions changed after 3 months. Within the initial 155 patients, 15% were offered a rehabilitation programme. The subsequent 127 patients were audited using three questions, demonstrating that 6% were referred, and 1% were enrolled into a rehabilitation programme, and 36% were educated regarding the benefits of exercise for HF. Discussion The experience throughout this audit was positive as it was flexible and well supported by the audit department. The electronic proforma is easily adaptable to incorporate subsequent clinical or research questions as desired. Treatment and monitoring reflect national recommendations, but rehabilitation referrals are below desired levels. A minority of patients have been referred to or enrolled in HF rehabilitation programmes reflecting poor national provision. Two-thirds of patients lived outside of London, making it impractical for them to frequently visit our hospital. Hence a hospital-based rehabilitation programme would not improve this care priority. Added questions demonstrated less than optimal numbers receive exercise education within clinic. Conclusion This audit demonstrated that a tertiary HF service has high rates of documentation, follow-up and compliance with established medical therapies. Notably many patients did not receive cardiac rehabilitation. This may be influenced by geographical limitations and the configuration of local services. To improve patient management further, alternative strategies including tele-health and enhanced multi-disciplinary team education are currently being explored.

Human embryonic stem cells (hESC) represent an essential source of cardiomyocytes for cardiac regeneration. Nuclear protein import (NPI) is critical in regulating cell differentiation and hypertrophy. The aim of this study was to determine if exposure of adult and hESC-derived cardiomyocytes (hESC-CM) to hypertrophic factor phenylephrine (PE) affects hypertrophy by altering NPI in comparison with adult failing rat and human cardiomyocytes. hESC-CM were generated by exposure of H7 hESCs to ActivinA and BMP-4. Failing cardiomyocytes were obtained from explanted human hearts at the time of transplant and from a rat model of myocardial infarction-induced hypertrophy and failure. hESC-CM and adult rat CM were exposed to PE for 48 h. The effects of P38 MAPK inhibitor, HDAC inhibitor, exportin-1 (CRM-1) inhibitor, and GSK-3β inhibitor were investigated by automated microscopy. Cell and nuclear sizes were increased in PE treated-adult rat cardiomyocytes (by 141±12.7% p

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.

Background Reperfusion arrhythmias can occur within seconds of restoration of blood flow to previously ischaemic myocardium. Heterogeneities and abrupt changes in both gap junctional coupling and action potentials during early reperfusion are thought to contribute to their pathophysiology. We tested the hypothesis that modulating gap junctional coupling can reduce the incidence of reperfusion arrhythmias, and used optical mapping to assess the mechanisms of any anti-arrhythmic effect. Methods Using a Langendorff apparatus, rat hearts were perfused with either 50 nM AAP10 to increase gap junctional coupling (n=10), 30 μM carbenoxolone (CBX) to reduce coupling (n=13) or control Krebs-Henseleit solution (n=10). Hearts were then subjected to regional ischaemia by occlusion of the left anterior descending artery for 8 min and then reperfused. Ventricular arrhythmia incidence was documented. For optical mapping experiments, a further eighteen hearts were loaded with a voltage-sensitive dye (RH237), perfused with an excitation-contraction uncoupler (10μM blebbistatin) and either AAP10 (n=6), carbenoxolone (n=6) or control (n=6), and then subjected to ischaemia and reperfusion as above whilst transmembrane voltage (V m ) transients were recorded. Results Both AAP10 and CBX reduced the incidence of reperfusion VF (AAP10 50%, CBX 77%, Control 90%) and delayed time to onset of reperfusion VF (AAP10 29.0±9.9 s; CBX 52.2±13.2 s; Control 21.8 s±6.4 s) (LogRank p 2 central ischaemic zone in the anterior left ventricle (LV) (Activation time: AAP10 15.3 ± 1.3ms; CBX 25.7 ±2.2 ms; Control 23.2±2.0 ms, p Conclusions Both increasing and reducing gap junctional coupling, with AAP10 and CBX respectively, reduced the rate and delayed the onset of reperfusion VF. This suggests that the partial uncoupling that occurs during early reperfusion is a determinant of reperfusion arrhythmias, and that both normalising the degree of coupling or further exacerbating uncoupling may be anti-arrhythmic. The anti-arrhythmic effect of increasing gap junctional coupling may be mediated by its dual effect of reducing action potential heterogeneity and improving conduction during reperfusion. Further uncoupling may be protective via attenuation of the abrupt changes in coupling and conduction that occur at reperfusion following ischaemia.

Sarcoplasmic reticulum calcium ATPase 2a (SERCA2a) gene therapy improves mechanical function in heart failure, and is under evaluation in a clinical trial. Restoration of sarcoplasmic reticulum (SR) Ca 2+ levels by SERCA2a gene therapy may alter SR Ca 2+ leak in the failing heart. A critical question is whether SERCA2a predisposes to increased SR leak and cellular triggered activity by restoring SR Ca 2+ levels? Methods Cardiomyocytes were studied isolated from rats with heart failure 4–6 weeks post in vivo AAV9SERCA (2×1011 drp) gene transfer (HF+S). Cells from untreated failing (HF) and non failing (NF) hearts served as controls. Spontaneous Ca 2+ spark generation was measured in fluo4-loaded cells by scanning confocal microscopy during a quiescent 30 s period after baseline stimulation. Field stimulated calcium transients were measured at 0.5 Hz, followed by caffeine application to measure SR Ca 2+ load. The ratio of sparks:SR load and Leak Index reflecting total SR Ca 2+ leak (spark frequency × amplitude × width × duration) were calculated. Baseline and isoproterenol (ISO)-induced triggered activity were measured in parallel cell studies, and ryanodine receptor (RyR) phosphorylation was measured. Results SERCA2a gene transfer normalised SR Ca 2+ load of failing myocytes (HF+S 5.86±0.64 vs HF 4.24±0.32 p 2+ transient relaxation kinetics (R50 (ms): HF+S 174±14, HF 292±42, NF 179±23 p Conclusion SERCA2a gene therapy reduced total SR Ca 2+ leak of failing myocytes whilst concurrently increasing SR Ca 2+ load to normal levels. Leak reduction was dependent upon altered spark characteristics as spontaneous spark frequency was unchanged, and reduced RyR phosphorylation supports resetting of SR Ca 2+ leak threshold after SERCA2a gene transfer. SERCA2a gene therapy reduced ISO-induced triggered activity in vitro and ISO-induced arrhythmias in vivo, and may represent a novel antiarrhythmic strategy in heart failure.

Authors' response to “Stress (Takotsubo) cardiomyopathy—a novel pathophysiological hypothesis to explain catecholamine-induced acute myocardial stunning”

Summary Arrhythmogenesis during heart failure is a major clinical problem. Regional electrical gradients produce arrhythmias, and cellular ionic transmembrane gradients are its originators. We investigated whether the nanoscale mechanosensitive properties of cardiomyocytes from failing hearts have a bearing upon the initiation of abnormal electrical activity. Hydrojets through a nanopipette indent specific locations on the sarcolemma and initiate intracellular calcium release in both healthy and heart failure cardiomyocytes, as well as in human failing cardiomyocytes. In healthy cells, calcium is locally confined, whereas in failing cardiomyocytes, calcium propagates. Heart failure progressively stiffens the membrane and displaces sub-sarcolemmal mitochondria. Colchicine in healthy cells mimics the failing condition by stiffening the cells, disrupting microtubules, shifting mitochondria, and causing calcium release. Uncoupling the mitochondrial proton gradient abolished calcium initiation in both failing and colchicine-treated cells. We propose the disruption of microtubule-dependent mitochondrial mechanosensor microdomains as a mechanism for abnormal calcium release in failing heart., Graphical Abstract, Highlights • Nanomechanical pressure application changes mechanosensitivity in failing heart cells • Microtubular network disorganization mediates the change in mechanosensitivity • Mitochondria are displaced from their original location and trigger calcium release • Uncoupling the mitochondrial proton gradient completely abolishes the phenomena, Miragoli et al. show that failing heart cells have altered sensitivity to nanomechanical stimuli mediated by changes in the alignment of microtubules. The microtubule network disorganization leads to displacement of mitochondria and alterations in calcium release.