Abstracts of the 2022 Autumn Meeting of the British Society for Cardiovascular Research ^†

Abstract

The Autumn Meeting of the British Society for Cardiovascular Research in 2022 was organized by David Grieve, Lauren Kerrigan, Claire Tonry and Chris Watson and held at the Wellcome-Wolfson Institute for Experimental Medicine, Queen’s University Belfast. The theme of the meeting was ‘Cardiac remodeling—basic mechanisms to clinical management’ and included an early career symposium. The Annual Bernard and Joan Marshall Distinguished Investigator Lecture was given by Professor Merry Lindsey on ‘Extracellular matrix remodeling in heart failure’. This paper presents the abstracts selected for oral and poster presentation.

1. Selected Oral Abstracts

1.1. Macrophage Subsets Differentially Regulate Cardiac Fibroblast Activation—Involvement of CXCL10/CXCR3 Signalling in Post-Myocardial Infarction Remodelling

• Georgios Kremastiotis ¹, Yong Li ², Alastair Poole ², Raimondo Ascione ¹, Jason L Johnson ¹ and Sarah J George ¹

¹ 

Medical School, Bristol Royal Infirmary, UK

² 

School of Physiology, Pharmacology and Neuroscience, University of Bristol, UK

Rationale: Post-myocardial infarction (MI) remodelling involves inflammatory and fibrotic processes. Pro- and anti-inflammatory macrophage-derived stimuli drive cardiac fibroblast (CF) activation. We hypothesized that distinct macrophage subsets differentially affect CF activation and aimed to identify macrophage regulators involved.

Methods and Results: In vitro, human primary monocytes were differentiated into anti-inflammatory (M-CSF) or pro-inflammatory (M-CSF + GM-CSF) macrophages. Human CFs exposed to anti-inflammatory macrophage secretome had significantly lower activation: decreased α-SMA expression (n = 7, 36%), proliferation (n = 6, 28%), migration (n = 4, 14%), and contraction (n = 4, 21%), compared to pro-inflammatory macrophage secretome. Proteomic analysis and Western blotting revealed IL1RN (log-fold-change = 2.55) and CTSZ (log-fold-change = 2.09) were significantly elevated in the pro-inflammatory macrophage secretome, while CXCL10 was significantly reduced (log-fold-change = 2.14). Gain-/loss-of-function experiments revealed CXCL10 reduced CF activation through CXCR3, while IL1RN promoted CF activation, and CTSZ-mediated effects were through CXCL10 degradation.

In vivo, MI was induced in C57BL/6J mice via permanent coronary artery ligation. Immunohistochemistry revealed abundant CXCL10, IL1RN, and CTSZ within macrophage-rich areas of infarcts at day 7 and their expression was significantly reduced by day 14 (n = 4; 85%, 89%, and 45%, respectively). The ratio of IL1RN+/CXCL10+ macrophages was significantly higher at day 7 (n = 3, 78%) compared to day 14, suggesting IL1RN/CXCL10 as surrogate markers of macrophage subsets. Moreover, significantly reduced CXCR3 expression in CF was detected at day 14 (n = 3, 57%).

Conclusions: Our findings demonstrate the divergent effects of distinct macrophage subsets and highlight the ability of CXCL10 to reduce CF activation. We propose intervention of the CXCL10/CXCR3 axis would be therapeutically beneficial in reducing maladaptive fibrosis in post-MI remodelling.

1.2. NOX4 NADPH Oxidase Signalling Is a Key Determinant of Angiogenic Function of Cord Blood Endothelial Colony-Forming Cells in Hypoxia

• Shun Hay Pun ¹, Karla M. O’Neill ¹, Hojjat Naderi-Meshkin ¹, Sudhir Malla ¹, Bianca Botezatu ¹, William King ¹, Philip D. Dunne ², Derek P. Brazil ¹, Chris J. Watson ¹ and David J. Grieve ¹

¹ 

Wellcome-Wolfson Institute for Experimental Medicine, UK

² 

The Patrick G Johnston Centre for Cancer Research, Queen’s University Belfast, UK

Objective: Myocardial infarction leads to adverse cardiac remodelling, driven largely by tissue ischaemia and hypoxia. Whilst cord-blood-derived endothelial colony-forming cells (CB-ECFCs) show clear potential to promote angiogenesis in post-infarction, their therapeutic application is limited by reduced efficacy in ischaemic tissue. This study aimed to define the specific impact of hypoxia in CB-ECFC-mediated neovascularisation.

Methods: CB-ECFCs (typically n = 9, 3 clones) were exposed to hypoxia (1% O²) or normoxia (21% O²) for 48h prior to analysis of angiogenic function (3D Matrigel assay), gene expression (qRT-PCR, Western blot, microarray), reactive oxygen species (H²O², DHE assay), and gene modification (plasmid overexpression [OE], siRNA knockdown [KD]).

Results: CB-ECFCs showed impaired tubulogenesis in hypoxia (tube area: normoxia 19.1 + 4.0, hypoxia 2.5 + 0.7 µm²; p < 0.001) which was associated with impaired metabolism (MTT, 59% vs. normoxia, p < 0.001) with maintained viability, and reduced expression of NOX4 and other functional genes (eNOS, HMOX1, VEGFR2), together with decreased H²O² (normoxia 0.46 + 0.03, hypoxia 0.08 + 0.02 arbitrary units; p < 0.001) and increased superoxide (normoxia 0.33 + 0.02, hypoxia 0.44 + 0.01 arbitrary units; p < 0.001). Parallel microarray analysis highlighted NOX4 as central to the altered signalling observed in hypoxic CB-ECFCs and identified PLAC8 as the upstream regulator. Whilst NOX4 OE partially restored angiogenic function in hypoxic CB-ECFCs (tube area: empty vector 18.7 + 2.1, OE 31.1 + 2.5 µm²; p < 0.05), PLAC8 KD reversed hypoxia-induced dysfunction (tube area: non-targeting control 5.6 + 2.0, KD 26.8 + 4.6 µm²; p < 0.01) with activation of NOX4 signalling.

Conclusions: Together, these findings highlight impaired NOX4 signalling as a key determinant of CB-ECFC angiogenic dysfunction in hypoxia, whilst PLAC8 may therefore represent a novel target to enhance NOX4-dependent vasoregenerative capacity with potential to reduce progression of adverse post-infarction cardiac remodelling.

1.3. Proteomics to Assess Myocardial Remodelling in Human Heart Failure and Explore the Effect of Mutations, Medications and Comorbidities

• Javier Barallobre-Barreiro ¹, T Radovits ², M Fava ¹, I Ragone ¹, L Daroczi ², L Ziani ¹, L Schmidt ¹, K Theofilatos ¹, MG Crespo-Leiro ³, N Domenech ³, B Merkely ² and M Mayr ¹

¹ 

James Black Centre, King’s College London, UK

² 

Semmelweis University Heart and Vascular Center, Budapest, Hungary

³ 

A Coruña University Hospital, Spain

Background. Our previous proteomics analyses demonstrated a profound effect of β-blockers on cardiac extracellular matrix composition in ischaemic heart failure (HF) patients. No comprehensive proteomics characterizations have been performed in non-ischaemic HF patients.

Methods. Using proteomics, we analyzed the left ventricular samples from ischaemic (n = 65) and non-ischaemic (n = 114) HF patients after heart transplantation, and non-failing controls (n = 19).

Results. The hallmarks of HF were detected in both HF groups, including elevated levels of atrial natriuretic peptides and the fibroblast marker vimentin, as well as decreases in creatine kinase M/B and troponin C. Compared to ischaemic HF, non-ischaemic patients showed elevated levels of proteins involved in proteasome activation. Moreover, despite similar clinical characteristics, responses to HF medications and the presence of comorbidities differed between HF groups. In ischaemic HF patients, β-blocker usage resulted in reduced proteoglycan deposition. In non-ischaemic patients, few changes were associated with medication usage, but hypertension was a critical determinant of myocardial protein remodelling. Among other changes, hypertensive HF patients showed a reduction in angiotensin-converting enzyme 2 compared to normotensive patients, and a reduction in the levels of CAVIN1-4, critical components of caveolae. Whole-exome sequencing demonstrated that in addition to comorbidities, pathogenic mutations determine proteomics profiles in non-ischaemic HF. Thus, proteins constituting the thick filaments were downregulated in carriers of titin truncating variants compared to patients with other mutations.

Conclusion. In the largest proteomics analysis of HF to date, we observed distinct protein remodelling processes in ischaemic and non-ischaemic HF patients and discerned the myocardial effects of mutations, medications and comorbidities.

1.4. Protection with LCZ696 (Sacubitril/Valsartan) against Diastolic Dysfunction and Cardiac Fibrosis in High-Fat Diet/Streptozotocin Mouse Model

• Narainrit Karuna ^1,2, Lauren Kerrigan ¹, Kevin Edgar ¹, Mark Ledwidge ³, Ken McDonald ³, David Grieve ¹ and Chris Watson ^1,3

¹ 

Wellcome-Wolfson Institute for Experimental Medicine, Queen’s University Belfast, UK

² 

Chiang Mai University, Chiang Mai, Thailand

³ 

STOP-HF Unit, St. Vincent’s University Healthcare Group, Dublin, Ireland

Introduction: Diabetic cardiomyopathy (DCM) involves complex pathophysiology with substantial changes at cellular and molecular level within the heart. Treatment options for DCM are limited; however, LCZ696 (sacubitril/valsartan), an angiotensin receptor neprilysin inhibitor (ARNI), may have therapeutic potential to improve diabetes-induced heart failure.

Objectives: We aimed to elucidate the cardio-protective effect of LCZ696 on diabetes-induced heart failure in a well-established mouse model of DCM.

Methods: DCM was induced in male C57BL/6J mice following a high-fat diet (HFD) and a single dose of 100 mg/kg streptozotocin (STZ). LCZ696 (100 mg/kg/day) or valsartan (50 mg/kg/day) was added to drinking water. Echocardiography was performed to evaluate left ventricle (LV) function and cardiac structure. Collagen I and III gene expression were measured by RT-qPCR.

Results: The DCM phenotype in the HFD/STZ mouse model was characterised by diastolic dysfunction and LV hypertrophy, along with preserved ejection fraction. Mice treated with LCZ696 for 12 weeks showed improvement in diastolic function and cardiac fibrosis, compared with the valsartan treatment (p < 0.05). The MV E/A ratio is the first diastolic parameter change after 4 weeks of LCZ696 treatment. Moreover, LCZ696 illustrated prevention of chronic progression of DCM by decreased left atrial volume and left atrial area. The cardiac fibrosis markers, collagen I and III, were suppressed by LCZ696 but not valsartan treatment. Neprilysin activity in the heart and plasma was inhibited by LCZ696 and comparable to control.

Conclusions: LCZ696 treatment improved diastolic dysfunction and cardiac fibrosis in the context of DCM. These beneficial effects may result from inhibiting the neprilysin enzyme which degrades several substrates related to cardiovascular diseases.

2. Selected Poster Abstracts

2.1. The Role of Small Nucleolar RNAs in Hypertrophic Cardiomyopathy

• Terri L Holmes ¹, Ben B Johnson ¹, Mandy J Peffers ², Chris Denning ³ and James GW Smith ¹

¹ 

University of East Anglia, UK

² 

University of Liverpool, UK

³ 

University of Nottingham, UK

Hypertrophic cardiomyopathy (HCM) is a heart disease affecting the structure, metabolism and contractility of cardiomyocytes. This can lead to serious clinical outcomes including sudden cardiac death. Small nucleolar RNAs (snoRNAs) are RNAs not translated into protein, instead contributing to various cellular functions including mRNA inhibition, chromatin modification and alternative splicing. SnoRNAs have been implicated in various metabolic and inflammatory diseases, but their role in cardiac diseases such as HCM has yet to be fully defined.

To investigate the involvement of snoRNAs in heart disease, human cardiomyocytes were differentiated from induced pluripotent stem cells (iPSCs) to generate 2D contractile heart tissue. These iPSC-CMs were subjected to cardiac stress conditions to observe the response in snoRNA expression. The snoRNA SNORD116 was found to have elevated expression levels under stress conditions. In addition, SNORD116 expression was shown to be elevated in the cardiac progenitor stages of cardiomyocyte differentiation. Overexpression of SNORD116 affected multiple pathways, crucial to cardiac signalling and function.

Overall, these results indicate that the snoRNA SNORD116 is an important modulator during cardiac development and disease. This work highlights the urgent need for further research into the role of snoRNAs in the context of cardiac health and disease. Importantly, snoRNAs may be exploited as targets in future RNA-targeting therapies.

2.2. Imaging Fibrosis in a Murine Model of Vascular Wall Injury Using Newly Developed Collagen Specific Imaging Agents

• Nadia Chaher ¹, Sara Lacerda ², Begoña Lavin ^1,3, Giuseppe Digilio ⁴, Julia Blower ¹, Rene Botnar ^1,5,6 and Alkystis Phinikaridou ^1,5

¹ 

School of Biomedical Engineering Imaging Sciences, King’s College London, UK

² 

Centre de Biophyisique Moléculaire, CNRS UPR 4301, Université d’Orléans, France

³ 

Department of Biochemistry and Molecular Biology, School of Chemistry, Complutense University, Madrid, Spain

⁴ 

Dipartimento di Scienze e Innovazione Tecnologica, Università del Piemonte Orientale, Vercelli, Italy

⁵ 

BHF Centre of Excellence, Cardiovascular Division, King’s College London, UK

⁶ 

Escuela de Ingeniería, Pontificia Universidad Católica de Chile, Santiago, Chile

Background: Excessive accumulation of extracellular matrix (ECM) proteins leads to tissue fibrosis which may result in organ failure. The two primary types of collagens that drive fibrosis in cardiovascular diseases are Collagen I and Collagen III (Col I and Col III). Current non-invasive methods to selectively image and quantify Col I and III are either limited or lacking. Here, we aimed to develop and preclinically validate collagen-specific probes to characterise tissue fibrosis by MRI.

Method: Newley developed collagen-specific probes containing a unique collagen-binding peptide, targeting Col I and III, were conjugated with a DOTA chelator enabling the co-ordination of lanthanides and radioisotopes. The probes were labelled with europium for in vitro binding assays to determine the dissociation constant (Kd); with 68Ga for in vivo PET/CT biodistribution studies and gadolinium for in vivo MRI of vascular remodelling following injury in a murine model.

Results: In vitro screening of the probes revealed that probe 1 was specific to Col III with a binding affinity Kd = 5.25 µM and no specific binding to Col I. Probes 2 and 3 showed binding to Col I and III but with specific and unspecific contributions. Biodistribution experiments showed favourable pharmacokinetics with fast blood clearance via renal excretion, negligible uptake in the liver and no unspecific uptake in other tissues. In vivo MRI using probe 1 allowed the detection of vascular remodelling in the diseased segment. Importantly, no enhancement was observed in the control segment. The results were validated histologically.

Conclusion: We have developed an imaging probe that targets collagen III that can enable the non-invasive detection, quantification and staging of fibrosis.

2.3. Glucose Metabolism Is Impaired in Cardiac Fibroblasts from Aged Rats Compared with Young Rats

• Kalyani Pandya ^1,2, Carlos J Alcaide-Corral ^1,2, Andrea Caporali ¹, Victoria Reid ^1,2, Mark G MacAskill ^1,2, Gillian Gray ¹ and Adriana AS Tavares ^1,2

¹ 

BHF Centre for Cardiovascular Science, UK

² 

Edinburgh Imaging; University of Edinburgh, UK

Within the myocardium, the extracellular matrix (ECM) is an integral component and collagen is the most abundant ECM protein involved in the maintenance of tissue structure and function through tightly regulated mechanisms. It has been suggested that glucose metabolism may play a role in cardiac collagen accumulation during natural aging through the formation of advanced glycation end-products (AGEs). AGEs can increase collagen crosslinking and myocardial stiffness leading to the development of cardiovascular disease. Sex-dependent left ventricular remodelling during natural aging has also been identified. In this study, we aim to explore the effect of age and sex on fibroblast glucose metabolism. Through a series of [¹⁸F]FDG in vitro cell incubation experiments, we have assessed glucose metabolism in primary rat cardiac fibroblasts from young (4 week) and aged (7 month) rats in the presence of D-glucose, L-glucose and deoxyglucose. Furthermore, we compared glucose metabolism differences between young (4 week) male and female rats. We have also conducted dose–response experiments with varying D-glucose concentrations (1–100mM). Preliminary data showed that age significantly reduces glucose metabolism in aged fibroblasts (mean ± SEM: 2.59 ± 0.26, p = 0.02) compared to young fibroblasts (3.9 ± 0.26). Sex appears to have no effect, at least in young animals. The D-glucose dose required to achieve 50% of inhibition (IC50) was found to be 25 mM, with maximum target engagement achieved at 50 mM in young cells. Age-dependent impaired glucose metabolism identified in this study could be an underlying mechanism fostering formation of AGEs, which in turn results in an adverse increase in collagen crosslinking in the myocardium.

2.4. Cardiac Injury Mirna Profile in Cancer Patients Treated with Doxorubicin

• David Mutigwa ¹, Michael Mallouppas ², Robin Chung ², Caroline Lozahic ¹, Helen Maddock ¹, J. Malcolm Walker ², Derek Yellon ² and Hardip Sandhu ¹

¹ 

Faculty Research Centre for Sport, Exercise and Life Sciences, Coventry University, UK

² 

The Hatter Cardiovascular Institute, University College London, UK

Anthracycline chemotherapy has improved the life expectancy of cancer patients, but despite its efficacy, it can lead to severe cardiotoxicity, which typically manifests as reduction in left ventricular ejection fraction, cardiomyopathy or symptomatic congestive heart failure. The early detection of subclinical cardiotoxicity is a challenge due to limited sensitivity and specificity of current clinical assessment tests. This project will focus on evaluating the expression levels of circulating cardiac-injury-associated miRNAs in doxorubicin-treated cancer patients. Total miRNA was extracted from pools of cancer plasma samples at baseline and post-chemotherapy with doxorubicin (i.e., immediately post-chemotherapy and 3 months post-chemotherapy). The differential expression pattern of cardiac injury-associated miRNAs was analysed by applying the pre- and post-chemotherapy pooled plasma samples on the Human TaqMan MicroRNA Array A cards. One third of the cancer patients treated with doxorubicin were affected by cardiovascular complications post-chemotherapy compared to pre-chemotherapy. Profiling of pooled plasma samples revealed a significantly altered expression of 21 miRNAs associated with cardiac injuries in the post-chemotherapy time points compared to pre-chemotherapy. The novel results from this study demonstrate that the expression levels of key cardiac injury-associated miRNAs are significantly altered after treatment with doxorubicin in cancer patients. In pending studies, we will investigate the key miRNA targets identified here on individual patient samples, to tease out the correlation between the severity of cardiac dysfunction with expression pattern of specific miRNAs. The identified panel of cardiac injury-associated miRNAs could potentially be used as screening biomarkers to assess cancer patients for early onset of subclinical cardiotoxicity.

2.5. Cardiac Injury miRNA Profile in Breast Cancer Patients Treated with Left-Sided Radiotherapy

• David Mutigwa ¹, Matthew Burrage ², Helen Maddock ¹, Vanessa M Ferreira ², David Cutter ³ and Hardip Sandhu ¹

¹ 

Faculty Research Centre for Sport, Exercise and Life Sciences, Coventry University, UK

² 

Oxford Centre for Clinical Magnetic Resonance Research, John Radcliffe Hospital, British Heart Foundation Centre of Research Excellence, UK

³ 

Oxford Population Health; University of Oxford, UK

Radiotherapy (RT) for breast cancer may cause incidental radiation exposure of the heart leading to radiation-related cardiac disease. Compared to right-sided RT, left-sided RT is linked to a higher risk of subsequent cardiac disease due to higher cardiac exposure from the left-sided treatments. Epidemiological studies have demonstrated a dose–response relationship between cardiac RT-dose and the later development of clinical disease. Early detection of subclinical cardiotoxicity is a challenge due to the limited sensitivity and specificity of clinical screening tests. This project focussed on evaluating the expression levels of circulating cardiac injury-associated miRNAs in RT-treated left-sided breast cancer patients. Baseline and four post-RT (24–72 h, 3–6 months, 6–12 months and 5 years post-RT) plasma samples from left-sided breast cancer patients treated with RT (incidental mean whole heart dose 1.40 ± 0.61 Gy) were analysed. Global miRNA was extracted from pooled plasma samples and analysed using the TaqMan Array Human MicroRNA A cards. Profiling of pooled plasma samples revealed a significantly altered expression of 27 miRNAs associated with cardiac injury in the post-RT time pools compared to the baseline pool. We hypothesise that the observed changes in expression levels of circulating miRNAs following RT treatment relate to the radiation exposure. Further studies investigating individual patient samples with the key miRNA targets identified are pending. These further assessments will enable miRNA signature changes to be related to individual variation in cardiac RT-dose. Identifying a panel of miRNAs could potentially be used as biomarkers to screen cancer patients for cardiac injury at an early stage.

2.6. Infarct Vessel Maturation Rather Than Angiogenesis Is a Feature of Infarct Repair Following Myocardial Infarction and Reperfusion in the Göttingen Mini-Pig

• Sara Al Disi ¹, Hinnah Imtiaz ¹, Raimondo Ascione ² and Gillian A Gray ¹

¹ 

Centre for Cardiovascular Science, Queens Medical Research Institute, University of Edinburgh, UK

² 

Translational Biomedical Research Centre, University of Bristol, UK

In the mouse experimental model of myocardial infarction, early angiogenesis during infarct repair leads to increased blood vessel density in the infarct and infarct border. Promotion of angiogenesis is considered to be a therapeutic target after MI, to enhance perfusion, reduce infarct expansion and therefore the likelihood of functional deterioration leading to heart failure. The aim of the present study was to characterise changes in myocardial blood vessel density in the translational Göttingen mini-pig following myocardial ischaemia and reperfusion (MI/R), induced by temporary balloon inflation in the left anterior descending coronary artery. Late-gadolinium enhancement MRI detected significant injury (13.7 ± 2.6% of LV) within 48h after MI/R, the ejection fraction determined by MRI 4 weeks after MI/R was 48.2 ± 2.7%, indicating functional decline. Masson’s trichrome staining confirmed infarct injury in fixed myocardial sections. Activated fibroblasts and macrophages indicated ongoing repair at 4 weeks after MI/R. In the remote myocardium, the majority of vessels immunopositive for the endothelial cell marker CD31 were <10 µm diameter (179 ± 31), consistent with the dominance of capillaries in the healthy tissue. In the infarct scar and border zones, the number of vessels <10 µm was reduced to 52 ± 8 (p < 0.01) and 105 ± 20 (p < 0.05), respectively, indicating vessel loss rather than neovascularisation. In contrast, the number of alpha-SMA + ve 10–100 µm vessels was increased in the scar (33 ± 4) relative to the remote (16 ± 1) zone. These data support vessel stabilisation by acquisition of a smooth muscle coat, rather than increased neovascularisation, as the primary change in myocardial vascularisation in the translational pig model during infarct repair, in contrast to the mouse.

2.7. Sex-Dependent Mitochondrial Function in Right Ventricle in an Experimental Model of Pulmonary Arterial Hypertension

• Chelbi Coyle, Margaret L MacLean and Lian Tian

• Strathclyde Institute of Pharmacy and Biomedical Science, University of Strathclyde, Glasgow

Background: Pulmonary arterial hypertension (PAH) has a female predominance with a ~4:1 female to male ratio. However, female PAH patients exhibit better right ventricular (RV) compensation in the face of increased pulmonary arterial pressures and thus better survival than the males. Though RV function is the major determinant of survival, the current therapies target the pulmonary vascular disease, rather than the RV directly. Impaired mitochondrial function has been shown to contribute to RV dysfunction, but its sex differences are unknown. This study aimed to determine the sex difference in mitochondrial function in RV in PAH in order to identify novel therapeutic targets.

Methods: Male and female Sprague Dawley rats were injected with Sugen (25 mg/kg) or vehicle and exposed to hypoxia for 3 weeks followed by 5 weeks in normoxia. RV haemodynamics were measured via pressure–volume loop measurement. Confocal imaging was performed on fresh RV to examine mitochondrial properties. Expression of protein was measured by Western blot.

Results: Sugen/hypoxia caused significant increases in RV systolic pressure and hypertrophy in both sexes, but reduced RV–pulmonary artery coupling in males only. Sugen/hypoxia caused significant decreases in mitochondrial membrane potential and mitochondrial fusion and a trend of decrease in mitochondrial superoxide in males only. The protein levels of mitochondrial transcription factor A and nuclear factor erythroid 2-related factor 2 were decreased in Sugen/hypoxia male RVs only, associated with impaired mitochondrial properties in males.

Conclusions: Mitochondrial function in RV displayed sex dimorphism in the Sugen/hypoxia rat model of PAH. Further investigations into the associated mitochondrial pathways could help unravel potential therapeutic targets for RV.

2.8. Cardiac Cachexia: Diagnostic Complications with Focusing on Weight Loss

• Donna Fitzsimons ¹, Loreena Hill ¹, Matthew A Carson ¹, Lana Dixon ², Patrick Donnelly ³, Susan E Piper ⁴, Gareth Thompson ¹, Theresa A McDonagh ⁴ and Joanne Reid ¹

¹ 

School of Nursing and Midwifery, Queen’s University Belfast, UK

² 

Royal Victoria Hospital, Belfast Health and Social Care Trust, UK

³ 

Ulster Hospital, South Eastern Health and Social Care Trust, Belfast, UK

⁴ 

Department of Cardiovascular Research, King’s College London, UK

Introduction: Cardiac cachexia is a syndrome that may occur in patients with advanced heart failure (HF); however, clinical identification remains challenging. Unexplained weight loss is often the first symptom reported by patients and their caregivers, warranting clinical investigation. This study explored the difficulties applying the cachexia diagnostic criteria of Evans et al. (2008) within a HF population.

Methods: A cross-sectional study was conducted in the Belfast Health and Social Care Trust, recruiting 200 patients with NYHA III-IV HF. Patients were assessed for cachexia based on the following: 5% weight loss in ≤12 months or BMI <20 kg/m² plus three of: (1) decreased muscle strength, (2) fatigue, (3) anorexia, (4) low fat-free mass index, and (5) abnormal biochemistry (elevated inflammatory markers (CRP and IL-6), anaemia (Hb < 12 g/dL) and serum albumin (<3.2 g/dL)).

Results: Oedema was present in 60.5% of the sample, with 30 patients (15%) identified with cardiac cachexia. The cachectic group had significantly (p < 0.05) lower BMI (21.8 vs. 29.9), but also fat-free mass index, muscle strength, red blood cell count and albumin; and significantly higher C-reactive protein (30.7 vs. 15.3), fatigue and anorexia issues.

Conclusions: Diagnostic criteria identified a 15% prevalence of cachexia within an advanced HF population living in Northern Ireland. Oedema can overshadow diagnosis within clinical practice. The results warrant further research to improve the clinical identification of the syndrome and aid development of a reliable assessment tool. Through early identification, tailored interventions could be implemented to improve patient care and outcomes.

2.9. One Size No Longer Fits All: Personalising Heart Failure Care

• Loreena Hill ¹, Donna Fitzsimons ¹, Lana Dixon ², David Thompson ¹, Claire McCune ², Anne McNulty ¹ and Chris Watson ¹ on behalf of the Passion-HF consortium

¹ 

Queen’s University Belfast, UK

² 

Belfast Health and Social Care Trust; Belfast, UK

Background: Heart Failure (HF) affects > 26 million people worldwide, with prevalence increasing due to ageing population. This additional burden on already overstretched healthcare resources, in staff and costs, is unsustainable. A change is warranted in how optimal healthcare is provided. Maximising patients’ self-care is mandatory; however, current HF-eHealth products are ‘add-ons’ to standard care, and can result in minimal benefits to patients or healthcare professionals.

Aim: To co-design an e-Health product to maximise the self-care of patients with heart failure.

Methods: The international PASSION-HF consortium, of which QUB is a partner, is developing an integrated eHealth-product enabling self-care and the self-prescription of medication. It includes novel features such as a decision support engine of treatment algorithms based on international HF guidelines integrated with self-learning AI algorithms, feedback systems and affiliated comorbidities. An interactive physician avatar interface and serious gaming tools will stimulate and improve compliance. Qualitative interviews with patients and caregivers have been conducted, with data informing development. The PASSION-HF prototype is currently being pilot tested for acceptability and proof of concept.

Effects: Data from interview with patients with HF and their caregivers (n = 82) identified the three following key themes: reassurance, personalised and transparency. The PASSION-HF next-generation eHealth product will enable personalised patient self-care. It will be easily accessible, providing support 24/7 with all decisions made available to the healthcare professional. Predicted results are a reduction of more than 70% of pressure on the health care system, nearly 50% less costs while improving patient care and potentially outcomes.

2.10. Exploring and Exploiting the Interleukin 11 Signalling Axis to Reduce Cardiac Fibrosis

• Michael Murray ¹, Leander Stewart ¹, Darren Tomlinson ² and Neil Turner ¹

¹ 

Discovery and Translational Science Department, Leeds Institute of Cardiovascular and Metabolic Medicine, School of Medicine, UK

² 

School of Molecular and Cellular Biology, Astbury Centre for Structural and Molecular Biology; University of Leeds, UK

Fibrosis is characterised by the overproduction of extracellular matrix proteins resulting in the impairment of cardiac contraction and relaxation, and interference of electrical signalling. Currently, there are no effective clinical treatments directly targeting fibrosis. The major cellular protagonists of tissue fibrosis are fibroblasts. In response to mechanical and pro-fibrotic stimuli, fibroblasts differentiate into a synthetically active myofibroblast phenotype. The recent evidence has suggested that the cytokine interleukin-11 drives a central unifying profibrotic signalling pathway in these cells, making it attractive for therapeutic exploitation. The first aim of this study is to characterise the relationship between IL-11 and the mechanosensitive ion channel Piezo-1 in mouse and human fibroblasts via the chemical and mechanical stimulation of Piezo-1 prior to measuring IL-11 mRNA and secretion. Secondly, novel affimer proteins (small antibody-like proteins) will be produced to interfere with IL-11 signalling. Affimer proteins which recognise IL-11 will be initially identified through sequential rounds of phage display. Purified affimers will then be tested for their ability to inhibit IL-11 signalling and attenuate fibrosis in cultured fibroblasts. The efficacy of affimers in inhibiting ERK phosphorylation will be used as a primary screen followed by analysis of more relevant fibrotic end points via collagen gel contraction assays, collagen staining and immunocytochemical techniques. Affimers which show the most promising results in vitro will then be investigated for their capacity to attenuate fibrotic cardiac remodelling in mice following chronic administration of angiotensin-II. These studies will pave the way for future therapeutic exploitation of the IL-11 signalling axis for reducing cardiac fibrosis.

2.11. Identification and Evaluation of Novel Protein Biomarkers for Atrial Fibrillation

• Claire Tonry ¹, Adam Russell-Hallinan ¹, Nadhezda Glezeva ^2,3, Patrick Collier ⁴, Ken McDonald ^2,3, Mark Ledwidge ^2,3, Ben Collins ⁵ and Chris J Watson ^1,2,3

¹ 

Wellcome-Wolfson Institute for Experimental Medicine, Queen’s University Belfast, UK

² 

HF Unit, St. Vincent’s University Healthcare Group, Dublin, Ireland

³ 

Heartbeat Trust, Dublin, Ireland

⁴ 

Department of Cardiovascular Medicine, Cleveland Clinic, Ohio, USA

⁵ 

School of Biological Sciences, Queen’s University Belfast, UK

Atrial fibrillation (AF) is linked with heart failure and is a primary cause of stroke. Routine monitoring of circulating biomarkers would significantly improve accuracy for prediction of AF and associated adverse events. The study cohort included (i) patients who developed new onset AF or paroxysmal AF, (ii) patients with pre-existing AF, who went on to develop either heart failure or have a stroke and (iii) age and sex match comparator groups who did not develop AF and/or any further MACE event over a similar time period. Serum samples collected at baseline, one year prior to event and at time of diagnosis, were analysed via mass spectrometry (MS) using the Evosep system on a Bruker™ TIMS-TOF instrument. Atrial appendage tissue (stored as FFPE) from an independent cohort of patients with AF (n = 10) and matched controls (n = 10) were also analysed. MS data were processed using Fragpipe and DIA-NN software. Significant proteins were identified using the “rstatix” package in R. The majority of significant serum protein changes associated with either AF/PAF/future stroke/future heart failure were observed at baseline, i.e., several years prior to diagnosis/event. The majority (71%) of significantly differentially expressed serum proteins were also detected in atrial appendage tissue. Twenty-one AF-associated proteins were significantly differentially expressed in both serum and tissue. The dataset has highlighted significant serum proteins associated with AF, PAF and risk of future heart failure/stroke. We have verified that these proteins are expressed in atrial tissue and may be pathologically relevant to the onset and progression of AF.

2.12. The Effects of Emetine Based Anti-Malarial Leads on Calcium Wave Dynamics in Sheep Ventricular Myocytes

• Matthew Jones ¹, Priyanka Panwar ¹, Natasha Hadgraft ², Niroshini Nirmalan ¹ and David Greensmith ¹

¹ 

Biomedical Research Centre, School of Science, Engineering and Environment, University of Salford, UK

² 

School of Healthcare Science, Manchester Metropolitan University, Manchester, UK

Anti-malarial drug resistance poses a major threat to world health, necessitating the need to develop alternative anti-malarial agents. Recent studies have shown the anti-amoebic drugs emetine dihydrochloride (EDC) and dehydroemetine (DHE) are effective as anti-malarial compounds. However, both are arrhythmogenic, compromising their clinical use. Our previous work showed that only DHE produced negatively inotropic alterations to Ca dynamics. However, both transiently increased systolic Ca on application, indicating ryanodine receptor (RyR) potentiation. To investigate this further, we examined the effects of EDC and DHE on intracellular Ca waves. Animals were killed in accordance with the Home Office Animal (Scientific Procedures) Act 1986. Sheep ventricular myocytes were loaded with the ratiometric Ca indicator Fura-2, then intracellular Ca waves were induced using 0.3mM ouabain octahydrate and 5mM extracellular Ca. The effects of EDC (50 nM) or DHE (80 nM) were measured using epi-fluorescent photometry. To estimate the SR Ca threshold for waves, we measured the amplitude of caffeine (10mM)-evoked Ca transients on wave onset. DHE decreased the Ca wave amplitude by 25 ± 6% (n = 20, p = 0.004) and increased the wave frequency by 10 ± 4% (n = 20, p = 0.02). The amplitude of the caffeine evoked Ca transient was decreased by 40 ± 12% (n = 9, p = 0.03) indicating a reduced SR Ca threshold for waves. EDC decreased the Ca wave amplitude by 19.83 ± 3% (n = 21, p = <0.001) but had no effect on the wave frequency or SR Ca threshold. These data suggest DHE potentiates the RyR; a phenomenon associated with arrythmia. However, it may be that the negatively inotropic effects of DHE previously reported offset those on the RyR reducing net arrhythmogenicity. 

2.13. Regulation of Dual Specificity Phosphatase (DUSP) Signalling by the Tsp1-Cd47 Axis in Hypertensive Heart Disease

• Zoe HR Haines, STE Cooper, SN Baldwin, GSTJ Whitley and DN Meijles

• St George’s University of London, UK

Introduction: Cardiac endothelial cell (CEC) dysfunction and fibrosis underscore cardiac remodelling in hypertensive heart disease (HHD). The mitogen-activated protein kinases (MAPKs) regulate these responses, which are selectively inactivated by DUSPs. Recently, the fibrotic matricellular protein thrombospondin 1 (TSP1) was shown to activate CEC MAPKs. Here, we determine DUSP abundance in hypertension and HHD, and identify TSP1-CD47 as a coordinator of DUSPs in CECs.

Methods: TSP1 and/or DUSP mRNA/protein expression was assessed in (1) human heart samples from non-ischemic failure vs. control (n = 12/12); (2) male spontaneously hypertensive rats (SHRs;11–13-wks) vs. age-matched Wistar controls (n = 6/6); (3) C57BL/6J male mice (10 wks) infused with vehicle or angiotensin-II (Ang II, 0.8mg/kg/d; n = 5–10) by osmotic minipump and (4) CECs isolated from wildtype mice, treated with the TSP1-peptide, 7N3 (10 μM; n = 4). Statistical tests: one way ANOVA with Holm–Sidak post-hoc test or unpaired t-tests.

Results: TSP1 abundance was elevated in human HF and Ang II mouse hearts. In human HF, DUSP5 (1.37 ± 0.13-fold, p = 0.0379) and DUSP8 (1.31 ± 0.18-fold, p = 0.0462) were elevated. In SHR hearts, DUSP7 (0.25 ± 0.07-fold, p = 0.0118) and DUSP6 (0.30 ± 0.10-fold, p = 0.0205) were reduced. In Ang II mouse hearts, DUSP1 (0.55 ± 0.06-fold, p = 0.350) and DUSP7 abundance was reduced (0.38 ± 0.14-fold, p = 0.0219), whilst DUSP6 was elevated (9.47 ± 6.77-fold, p = 0.0470). In CECs, 7N3 elevated DUSP1 (2.96 ± 0.64 fold), DUSP5 (1.84 ± 0.17 fold) and DUSP8 (2.41 ± 0.49 fold) expression.

Conclusion: The initial response to hypertension alters nuclear MAPK activity (via DUSP1), whilst progression to HHD and failure is controlled by DUSP5-8 (via inhibited ERKs). Altered DUSP5/8 expression in CECs may underscore overall maladaptive remodelling. Here, we identify the TSP1-CD47-DUSP axis as a strategy to ameliorate HHD progression to HF.

2.14. Myofibroblast-Specific Genetic Deletion of Piezo1 Protects against Hypertension-Induced Cardiac Hypertrophy

• Leander Stewart, T Simon Futers, Fiona Bartoli, Mark Drinkhill, David Beech and Neil Turner

• Leeds Institute of Cardiovascular and Metabolic Medicine, University of Leeds, UK

Piezo1 is a mechanosensitive non-selective cation channel that is expressed in both cardiomyocytes and cardiac fibroblasts. While little is known about the functional mechanisms of Piezo1 channel activity within the heart, the genetic deletion of Piezo1 in cardiomyocytes has been described as preventing pressure overload-induced hypertrophy. The contribution of cardiac fibroblast Piezo1 to the pathological cardiac environment is unknown. We have previously discovered that the in vitro activation of cardiac fibroblast Piezo1 drives p38 MAP Kinase phosphorylation and the upregulation of IL-6 expression and secretion, indicating a role for cardiac fibroblast Piezo1 signalling in the response to cardiac pathology. To investigate the in vivo role of activated cardiac fibroblast (myofibroblast)-specific Piezo1 signalling in hypertension-induced cardiac remodelling, we employed a Postnmcm;Piezo1fl/fl mouse model which drives an inducible deletion of Piezo1 specifically in myofibroblasts. Postnmcm;Piezo1fl/fl mice were challenged with 28-days of subcutaneous angiotensin II infusion, to stimulate the myofibroblast phenotype and promote pressure-induced cardiac hypertrophy. The cardiac hypertrophic response to angiotensin II was determined through biometric analysis, echocardiography, and RT-qPCR measurements of gene expression. Biometric and echocardiographic analysis demonstrated that myofibroblast-specific genetic deletion of Piezo1 reduces cardiac hypertrophy and helps maintain normal cardiac function after chronic angiotensin II infusion in 12 week old mice. In summary, our results demonstrate that cardiac fibroblast-specific Piezo1 contributes to the hypertrophic response of the heart to hypertension.

2.15. Anthracyclines Increase Oxidative Stress in Sheep Ventricular Myocytes

• Amy Foster, Courtney Riley and David J Greensmith

• Biomedical Research Centre, School of Science, Engineering and Environment, The University of Salford, Manchester, UK

Anthracyclines are effective chemotherapeutics but are associated with adverse cardiac outcomes in cancer survivors. Whether increased production of reactive oxygen species provides a cardiotoxic mechanism remains unclear. To elucidate this, we developed a method to measure anthracycline-evoked oxidative stress in cardiac myocytes. Primary sheep ventricular myocytes were isolated in accordance with the Animal (Scientific Procedures) Act 1986. Cells were treated with 0.001 to 10 μM doxorubicin (DOX) or daunorubicin (DAUN) for 30 min, before loading with CellROX red at 5 μM for 30 min. Fluorescent and brightfield images of cells were captured using the Biotek Cytation 3 imaging system. Cell fluorescence was quantified off-line using ImageJ. To test the methodology, cells were exposed to 100 μM hydrogen peroxide which increased fluorescence by 44.3 ± 15.7% (n = 10, p < 0.05). DOX produced a concentration-dependent increase in fluorescence (n = 22, p < 0.05) while DAUN only increased fluorescence at 0.001 and 10 μM (n = 19, p < 0.05). To determine the time-dependent effect, we exposed cells to 0.1 μM DOX and DAUN for 240 min. Both increased oxidative stress within 10 min (n = 19 and 17, p <0.05) which persisted over the experiment’s time course. These data suggest both DOX and DAUN can elevate oxidative stress in cardiac myocytes. Our next experiments seek to elucidate the intracellular mechanisms. Furthermore, we provide a useful method to measure oxidative stress in cells.

2.16. The Role of Interferon Signalling Pathway in the Pathogenesis of Experimental Diabetic Cardiomyopathy

• Mohammed Alsaggaf, Lauren Kerrigan, Kevin S Edgar, Narainrit Karuna, Karla M O’Neill, Shun Hay Pun, Oisin Cappa, Chris J Watson and David J Grieve

• Wellcome-Wolfson Institute for Experimental Medicine, Queen’s University Belfast, UK

Objective: Diabetic cardiomyopathy (DCM) is a major complication of type 2 diabetes that is specifically linked to coronary endothelial cardiac remodelling and heart failure. While many pathological pathways are implicated to DCM, however specific signalling mechanisms are still poorly defined. This study aimed to determine the differentially regulated signalling pathways in experimental DCM with the endothelial cell enrichment that underlies microvascular dysfunction.

Methods: Experimental DCM was induced in adult male C57BL6/J mice (n = 4) by high-fat diet (5450 kcal/kg) and single-dose streptozotocin (100 mg/kg i.p.) with comparison to chow-fed vehicle-injected controls. Mice were sacrificed at 6 months and left ventricular tissue was isolated for RNA sequencing and bioinformatics analysis.

Results: R and Partek were used to generate two lists of genes differentially expressed between DCM and control hearts (adjusted p-value 0.05, log2 fold change −1.5 to +1.5), which were filtered for enrichment in capillary endothelial cells based on a published human cardiac cell atlas (Litviňuková et al. Nature 2020; 588: 466–472). This analysis identified 619 genes with significant endothelial expression, which were uploaded to Ingenuity Pathway Analysis (IPA) to interrogate conical pathways associated with microvascular dysfunction in the diabetic heart. The most significantly altered conical pathways were interferon and interferon regulatory factor signalling. Interestingly, interferon signalling has been associated to endothelial dysfunction, but not in the diabetic heart.

Conclusion: Endothelial interferon signalling may represent a key driver of coronary microvascular dysfunction in experimental diabetes. The validation of important targets within related pathways can lead to new treatment approaches.

2.17. Investigating the Regulation of Human Cardiac Fibroblast Behaviour by Micro-Rna-214 in the Context of Pathological Cardiac Remodelling

• Christopher J Trevelyan, L Stewart, KE Porter, KA Forbes and NA Turner

• Leeds Institute of Cardiovascular and Metabolic Medicine, School of Medicine, University of Leeds, UK

Background: Cardiac fibroblasts (CFs) regulate cardiac remodelling through extracellular matrix (ECM) turnover and stimulation of cardiac hypertrophy. CFs are activated by cytokines and mechanical stimuli (detected by mechanosensitive ion channels such as Piezo1). Our group found that isoproterenol infusion in mice increased miR-214-3p expression (dependent upon p38α MAPK). Our aim is to understand the role miR-214 plays in CF behaviour during remodelling.

Method: Human CFs were isolated from right atrial appendage biopsies. Pre-miR-214-3p was transfected for overexpression and anti-miR-214-3p for inhibition. mRNA expression levels were measured by qRT-PCR and protein expression by Western blotting. Tandem mass tagging (TMT) proteomics was performed to quantify differential protein expression. Bioinformatics tools such as Ingenuity Pathway Analysis were used to identify targets for downstream functional analysis.

Results: Out of 8793 proteins, 3387 were significantly changed at 72 h post-pre-miR-214 transfection. Bioinformatics analysis identified two distinct protein networks associated with miR-214, with mitochondrial dysfunction being the most significantly modulated. A particular decreased mitochondrial protein of interest was mitofusin-2, a fusion protein proposed as a repressor of the lysyl oxidase family of collagen cross-linking enzymes, which showed a concomitant increase in our proteomics screen. We also discovered that pre-miR-214 transfection led to a ~50% decrease in Piezo1 (mRNA and protein), a mechanosensitive cation channel activated in response to mechanical tension, leading to calcium influx, p38 MAPK activation and IL-6 release.

Conclusion: Understanding the downstream pathways regulated by miR-214 could identify novel therapeutic targets in the treatment of heart failure.

2.18. Modulation of Tetranectin Expression Impacts Cardiac Fibroblast Function and Is Associated with Changes in Cardiac Fibrosis In-Vivo

• Kevin Edgar, Lauren Kerrigan, Adam Russell-Hallinan, Lucy Burrows, David Grieve and Chris Watson

• Wellcome-Wolfson Institute for Experimental Medicine, Queen’s University Belfast, UK

Heart failure is a major public health concern which occurs due to impairment in function and structural changes that arise in the heart following various insults, including ischaemia, hypertension and diabetes. Heart failure is characterised by increased fibrosis, cardiomyocyte hypertrophy, inflammation and microvascular remodelling in the heart. Tetranectin is found at high levels in normal serum and in some tissues during development, but not in normal adult tissues. It is important in extracellular matrix remodelling (ECM), which is particularly important in heart failure, due to its ability to bind ECM components and has been suggested to have a protective function within the muscle, bone, and the circulatory system. The changes in the myocardium leading to heart failure heavily involve ECM remodelling and by using mice deficient in tetranectin we wanted to investigate this in more detail. A model of angiotensin II-mediated cardiomyopathy was induced in tetranectin knockout mice by subcutaneous angiotensin II infusion for 4 weeks. Cardiac function was assessed in animals by echocardiography and heart tissue was collected for analysis of fibrosis and hypertrophy by gene and protein expression and histological analysis to investigate structural changes. There was increased blood pressure along with some evidence of cardiac dysfunction following angiotensin infusion. There was significant hypertrophy and fibrosis with some differences in fibrotic gene expression induction between wild-type and knockout mice. This study demonstrates that angiotensin II infusion can cause cardiac changes associated with HF and that knockout of tetranectin can modulate the degree of cardiac dysfunction and fibrosis.

2.19. The Transcriptomics Characterization of a PMCA1 Cardiomyocyte-Specific Knockout Mouse Model

• Alexandru Chelu, Elizabeth J Cartwright and Bernard Keavney

• University of Manchester, UK

Heart failure is the endpoint of many cardiovascular diseases and represents a leading cause of death worldwide. Plasma membrane calcium ATPase 1 (PMCA1) is a calcium-extruding pump found in virtually all cells. Several studies have linked PMCA1 to hypertension, myocardial infarction and heart failure, and it has been proposed as a therapeutic target against heart failure. PMCA1 has an important signalling role in the heart but this function is poorly understood. Using a Cre-recombinase strategy, we generated a PMCA1 cardiomyocyte-specific knockout (PMCA1CKO). To investigate the signalling role of PMCA1 in the heart, we characterised the transcriptome of ventricular tissue from PMCA1CKO mice via RNA-sequencing. Differential expression analysis revealed significant upregulation of 316 genes and downregulation of 274 genes (FDR < 0.05). The dilated cardiomyopathy signalling pathway was the most enriched canonical pathway, followed by the eNOS, nNOS and NFAT signalling pathways. Using toxicity annotations, 45 genes were associated with seven phenotypes of heart failure including cardiac enlargement, fibrosis and dilation. Furthermore, 14 ion channels were differentially expressed in PMCA1CKO and interestingly 5 of these were linked to arrhythmia, heart failure or both. The altered gene expression of candidate heart failure genes identified in silico such as SPRR1A, NPPA, CACNB1, CACNA1S and ANKRD1 was further confirmed experimentally. Taken together, these results suggest that PMCA1 may play a role in heart failure onset by regulating key signalling processes linked to cGMP and cAMP pathways. Furthermore, altered expression of ion channels are consistent with previous work in our lab demonstrating a link between PMCA1 and arrhythmia.

2.20. Collagen Signalling Pathways in the Progression of Hypertensive Heart Disease

• Hazel Blythe ¹, Katie Skeffington ¹, Megan Young ¹, Saadeh Suleiman ¹, Emma Hart ², Angus Nightingale ^2,3 and Ana Abdala Sheikh ²

¹ 

Bristol Medical School

² 

School of Physiology, Pharmacology and Neuroscience, University of Bristol, UK

³ 

Bristol Heart Institute, Bristol Royal Infirmary, University of Bristol, UK

Hypertensive heart disease is a multifactorial disease characterised by molecular, structural and functional abnormalities of the left ventricle (LV). Myocardial fibrosis, a common feature of hypertension, results from excessive collagen deposition, and increases the risk of cardiovascular morbidity and mortality. To address this problem, we used spontaneously hypertensive (SHR) male rats and Wistar Kyoto (WKY) controls to identify disease-associated functional, structural and molecular changes. Cellular signalling pathways associated with fibrosis will be studied using proteomics to provide insights into the temporal progression of myocardial fibrosis in hypertension. Echocardiography was conducted at 10- and 15-weeks old, prior to tissue collection. Ejection fraction and fractional shortening were similar at 10-weeks old for SHR and WKY; however, both were lower at 15-weeks old in SHRs compared to WKYs (p = 0.0103 and p = 0.0022, respectively). Hearts were collected from SHR and WKY at 10- (n = 6) and 15-weeks old (n = 4) for each group. Hearts were fixed and stained to assess structure and fibrosis. Masson’s staining revealed similar levels of interstitial fibrosis at 10-weeks old between SHRs and WKYs. At 15-weeks old, interstitial fibrosis was significantly higher in SHRs in the interventricular septum (p = 0.0265), LV free wall (p = 0.0325) and LV posterior wall (p = 0.0196), compared to WKYs. Histological analyses revealed hypertrophy of the LV anterior wall in SHRs compared to WKYs (p = 0.0112), in addition to a higher relative wall thickness in SHRs (p = 0.0263). Proteomics analyses are ongoing and may provide mechanistic pathways via which cardiac hypertrophy and fibrosis is driven in hypertension, leading to identification of potential therapeutic targets.

2.21. Quaking-7: A Targetable Mediator of Diabetic Vasculopathy

• Victoria A Cornelius ¹, Hojjat Naderi-Meshkin ¹, Shu-Dong Zhang ², Alan Stitt ¹, David Grieve ¹ and Andriana Margariti ¹

¹ 

Wellcome-Wolfson Institute for Experimental Medicine, Queen’s University Belfast, UK

² 

Northern Ireland Centre for Stratified Medicine, Ulster University, Londonderry, UK

Despite the high morbidity, mortality and economic burden, the mechanisms behind diabetic vascular dysfunction remain predominantly unclear, limiting the available treatment strategies. Using stem cell technologies, we recently discovered the RNA-binding protein Quaking-7 (QKI-7) to be significantly upregulated in diabetic endothelial cells, greatly impairing the endothelial cell barrier, compromising angiogenesis and enhancing monocyte adhesion. Moreover, the knockdown of QKI-7 in vivo, using a hindlimb ischemia diabetic mouse model, resulted in significant reperfusion and blood flow recovery, highlighting its potential as a therapeutic target. To uncover the pathogenic mechanism of QKI-7, RNA-immunoprecipitation sequencing was performed on diabetic and non-diabetic iPS-ECs. A total of 42 RNAs were found to be statistically significantly enriched by QKI-7. Many of which, through IPA analysis, were found to have fundamental roles in both the vascular development and vascular disease onset networks as well as be affiliated with 48 pathways connected to vasculature health. The overexpression of QKI-7 in ECs, as well as the analysis of diabetic iPS-ECs, confirmed QKI-7 as significantly regulating the expression of these enriched RNAs. Through direct gene regulation, QKI-7 was therefore found to mediate a vast network of endothelial cell dysfunction, contributing to the progression of vascular disease. To therapeutically target QKI-7, connectivity mapping was performed. The mapping of specific gene signatures against libraries of FDA-approved drugs revealed compounds with the theoretical potential to inhibit QKI-7. Analysis of diabetic iPS-ECs confirmed the compounds ability to significantly reduce QKI-7 expression as well as restore vascular function. Manipulation of QKI-7 therefore represents a promising strategy for the treatment of diabetic vascular complications.

2.22. Do Plasma Levels of Interleukin-6 and 10 Correlate to Cardiac Dysfunction in Coronary Artery Disease?

• Bethan Samphire-Noden ¹, Anna Borun ¹, Courtney Riley ¹, Matthew Jones ¹, Sarah Withers ^1,2, Vasanthi Vasudevan ³, Mohamad Nidal Bittar ³ and David Greensmith ¹

¹ 

Biomedical Research Centre, School of Science, Engineering and Environment, University of Salford, UK

² 

Salford Royal Foundation Trust, UK

³ 

Lancashire Cardiac Centre, Blackpool Victoria Hospital, UK

Coronary artery disease (CAD) is a leading cause of morbidity and mortality in the United Kingdom. In CAD, inflammation is a key pathogenic factor and elevated levels of inflammatory markers such as cytokines are associated with decreased cardiac function. However, the clinical studies examining this are limited. In this preliminary study, we measured plasma levels of a pro- and anti-inflammatory cytokines in a CAD patient cohort then correlated the levels to indices of cardiac function. This study was conducted in accordance with local and IRAS ethical approval (IRAS ID: 247341). Preoperative serum samples were collected from patients scheduled for routine coronary revascularisation surgery. Serum interleukin-6 (IL-6) and interleukin-10 (IL-10) concentrations were measured using high-sensitivity ELISA kits (Abcam, Cambridge, UK; Invitrogen, Waltham, MA, USA). Average IL-6 and IL-10 concentrations were 16.02 ± 0.01 pg/mL and 4.06 ± 0.01 pg/mL, respectively. Interestingly, IL-6 and IL-10 levels did not correlate with LVOT obstruction or ejection fraction in our patient cohort (n = 16–31). However, IL-6 levels significantly correlated with pulmonary arterial systolic pressure (PASP) (n = 8, p = 0.03), end systolic volume (ESV) (n = 9, p = 0.01) and end diastolic volume (EDV) (n = 9, p = 0.02). IL-10 was only significantly correlated with ESV (n = 11, p = 0.02). These preliminary data indicate that IL-6 and IL-10 may be of use as diagnostic markers in CAD patients. We now aim to increase the study power and identify key correlates.

2.23. Doxorubicin-Induced Vasotoxicity in Coronary Vessels: Investigating the G-Protein Coupled Receptor Mediated Vasoconstriction and the Effect of an MEK 1/2 Inhibitor U0126

• Caroline Lozahic, Mark Wheatley, Helen Maddock and Hardip Sandhu

• Faculty Research Centre for Sport, Exercise and Life Sciences, Faculty of Health and Life Sciences, Coventry University, UK

Doxorubicin (Dox), an efficient chemotherapeutic drug, is cardiotoxic. Vascular stress can lead to increased G-protein coupled receptor (GPCR)-mediated vasoconstriction through binding of specific GPCR ligands, such as the 5-Hydroxytryptamine 1B (5-HT1B) receptor and thromboxane A2 (TXA2) receptor. The activation of those two receptors leads to the signalling through the MEK1/2 pathway. This study used an organ culture model to investigate the GPCR-mediated vascular tone in Dox-treated coronary vessels, and a potential adjuvant MEK1/2 inhibitor U0126. Coronary arteries from rats were dissected into four groups (Vehicle, 0.5 µM Dox, 0.5 µM Dox with 5/10 µM U0126, and 5/10 µM U0126). Dose–response curves of the 5-HT1B receptor agonist and TXA2 agonist were obtained by wire-myography after 24 h of organ culture in DMEM ± 0.5 µM Dox ± 5 µM U0126. Furthermore, 5-HT1B and TXA2 mRNA expression in LAD were investigated by qRT-PCR. The 5-HT1B dose–response curve was significantly increased by incubation with Dox at specific doses and 5-HT1B mRNA expression was significantly increased in presence of 0.5 µM Dox compared to vehicle (p < 0.05). Incubation with 0.5 µM Dox increased the vasocontraction through TXA2 significantly at specific doses, while co-administration of 10 µM U0126 with 0.5 µM Dox showed a decrease in TXA2-mediated vasoconstriction compared to 0.5 µM Dox mono-treatment at 10-6.5M U46619 (p < 0.05). This study shows that the expression of 5-HT1B and TXA2 is altered during Dox-induced cardiotoxicity. Further studies unravelling the involvement of GPCR pathways and mediators will broaden our understanding of Dox-induced vasotoxicity, and the potential of the MEK1/2 inhibitor as an adjuvant therapy option.

2.24. The Role of Inflammation and Oxidative Stress in Coronary Artery Disease

• Courtney Riley ¹, Bethan Samphire-Noden ¹, Anna Borun ¹, Matthew Jones ¹, Vasanthi Vasudevan ², Nidal Bittar ², Sarah Withers ^1,3 and David Greensmith ¹

¹ 

Biomedical Research Centre, School of Science, Engineering and Environment, University of Salford, UK

² 

Lancashire Cardiac Centre, Blackpool Victoria Hospital, UK

³ 

Salford Royal Foundation Trust, UK

Coronary artery disease (CAD) is a leading cause of death worldwide. The primary pathological mechanism is atherosclerosis, an inflammatory process associated with increased cytokine levels and oxidative stress (OS). However, it is not clear to what extent either are elevated in CAD patients. This preliminary study sought to measure plasma levels of interleukin-1β (IL-1β), and oxidised-low density lipoprotein (Ox-LDL—a marker of OS) in a CAD patient cohort, then correlated the values to indices of cardiac function. This study was conducted in accordance with IRAS ethical approval (ID: 247341). Preoperative plasma IL-1β and Ox-LDL levels were quantified using ELISA (IL-1β; ThermoFisher Scientific, UK; Ox-LDL; Abcam, UK). Indices of cardiac function were acquired from patient echocardiography records. IL-1β and Ox-LDL concentrations were 1.46 ± 0.26 pg/mL (n = 31) and 32.76 ± 6.15 ng/mL (n = 37), respectively. Peak E wave velocity negatively correlated with plasma IL-1β concentration (n = 13, R² = 0.4506, p = 0.0120). Tricuspid annular plane systolic excursion (TAPSE) positively correlated with plasma Ox-LDL levels (n = 15, R² = 0.3067, p = 0.0322). Other indices of systolic function such as ejection fraction did not correlate with either IL-1β or Ox-LDL. Our preliminary findings suggest that IL-1β and Ox-LDL may be useful biomarkers to assess cardiac function in CAD. Future work will increase the study power and elucidate the underlying oxidative mechanisms.

2.25. Preconditioning with Alpha-1A Receptor Agonist A61603 Is Cardioprotective against Global Ischemia and Reperfusion Injury in Healthy Hearts, but Not in Failing Rat Hearts

• Megan Young, Amy Harris, Katie Skeffington, Saadeh Suleiman and Massimo Caputo

• Bristol Medical School, University of Bristol, UK

Infarcted hearts undergo significant remodelling into failure which can involve signalling pathways responsible for cardioprotection. α1A-adrenergic receptors which are present on 60% of cardiomyocytes have been implicated in cardioprotection in normal hearts. The stimulation of these receptors produces an inotropic response and activates myocyte survival signalling pathways. However, whether this cardioprotection can also be seen in diseased hearts is not presently known. This study aimed to investigate whether preconditioning with A61603, a selective α1A agonist, is protective against global ischaemia/reperfusion injury (IRI) in both healthy and diseased adult male rat hearts. Acute infarction was induced by occlusion of the LAD. Echocardiography measurements after 4 weeks confirmed a reduction in cardiac function with the ejection fraction reduced to 44%. Both healthy and failing rat hearts were cannulated on a Langendorff setup and subjected to 30 min ischemia and 60 min reperfusion, with/without prior A61603 treatment (10 nM). To assess cardioprotection, the haemodynamic functional recovery and lactate dehydrogenase release were measured throughout reperfusion. The extent of injury in the myocardium was determined by triphenyltetrozolium chloride staining at the end of reperfusion. The treatment with A61603 increased the left ventricular developed pressure, in both healthy and failing hearts, and this effect was reversed by the washout period. Pre-treatment with A61603 improved recovery and reduced injury in healthy hearts. In contrast, A61603 did not confer protection against IRI in failing hearts. This work demonstrates that targeting the α1A-adrenergic receptors is cardioprotective against global ischemia reperfusion injury in healthy, but not failing hearts. The underlying signalling pathways responsible require further investigation.

2.26. Reduced Pro-Angiogenic Function of Endothelial Colony-Forming Cells in Hyperglycaemia Is Mediated by NOX4

• Bianca Botezatu ¹, Tinrui Toh ¹, David C Campbell ¹, Kevin S Edgar ¹, Jyoti Kandel1 ¹, Arya Moez ¹, Eleanor K Gill ¹, Rawan A Abudalo ¹, Xin N Wong ¹, Catherine McClintock ¹, Salman Ashraf ¹, Shun H Pun ¹, Mervin C Yoder ², Alan W Stitt ¹, Reinhold J Medina ¹, David J Grieve ¹ and Karla M O’Neill ¹

¹ 

Wellcome-Wolfson Institute for Experimental Medicine, Queen’s University Belfast, UK

² 

Department of Pediatrics, Indiana University School of Medicine, Indianapolis, Indiana, USA

Objective: Cord-blood-derived endothelial colony-forming cells (CB-ECFCs) are a defined progenitor subtype with established roles in vascular homeostasis and angiogenesis. They possess low immunogenicity and show allogeneic or autologous therapeutic potential for the improved management of ischaemic cardiovascular disease, whilst targeting key angiogenic signalling pathways remains a key focus for enhancing CB-ECFC intrinsic function. The emerging evidence indicates that CB-ECFCs are regulated by NADPH oxidase (NOX)-derived reactive oxygen species (ROS), with their angiogenic capacity negatively impacted by hyperglycaemia. The aim of this study was to investigate the specific influence of CB-ECFC NOX-dependent ROS signalling in experimental diabetes.

Methods: CB-ECFCs (n ≥ 3, up to 4 clones) were cultured in high glucose (DG, 25 mmol/L) or normal glucose control (CTL, 5 mmol/L) for 72 h, with or without PMA stimulation (500 nmol/L), prior to analysis of ROS generation (DHE fluorescence), angiogenic function (Matrigel assay), gene expression (qRT-PCR) and impact of NOX4 plasmid overexpression (OE).

Results: DG treatment increased CB-ECFC ROS generation and prevented PMA-induced angiogenesis. Notably, the upregulation of NOX4 mRNA expression was observed in PMA-treated CB-ECFCs under CTL conditions, but this response was lost with DG culture, suggesting an important role for NOX4-derived ROS. Indeed, NOX4OE completely rescued the pro-angiogenic response of CB-ECFCs after DG treatment both basally and with PMA stimulation to levels above those observed in CTL.

Conclusions: These data indicate that CB-ECFC angiogenic dysfunction in hyperglycaemia may be mediated by reduced NOX4 signalling, thus highlighting this major NOX isoform as a potential target to enhance the vasoreparative capacity of CB-ECFCs in diabetes.

2.27. A Semi-Automated Method to Evaluate Microvascular Physiology in the Conjunctival Microcirculation

• Julie S Moore ^1,2, Min Jing ³, Jonathan A Mailey ⁴, Agnes Awuah ¹, Paul F Brennan ⁴, Louise J Robertson ^1,2, William P Burns ³, M Andrew Nesbit ^1,2, James AD McLaughlin ^2,3, Mark S Spence ⁴ and Tara Moore ^1,2

¹ 

School of Biomedical Sciences, Ulster University, UK

² 

Integrated Diagnostics Laboratory, Ulster University, UK

³ 

Nanotechnology and Integrated BioEngineering Centre, Ulster University, UK

⁴ 

Royal Victoria Hospital, Belfast Health and Social Care Trust, UK

Introduction: Cardiovascular disease is associated with both structural and functional alterations and remodelling within the microcirculation. Coronary microvascular dysfunction has previously shown to be prognostically adverse. The conjunctival microvasculature is accessible for non-invasive imaging. Smartphone applications are increasingly utilised for the diagnosis and long-term monitoring of systemic conditions. The aim of this study was to develop an application for the non-invasive assessment of the conjunctival microcirculation.

Methods: A slit-lamp (magnification ×40) was used in combination with an iPhone 11 Pro (2x zoom). A fixation point was used to help patients focus, and hence obtain a more stable video. For each participant, the left temporal, left nasal, right nasal and right temporal views of the front of the eye were imaged. Videos of ~10 s duration, were reviewed prior to processing, and videos with excessive eye movement were repeated. Sixty continuous focused frames with minimal eye movement were selected for analysis. Image registration, vessel-filtering, centreline extraction, branch point detection, diameter and velocity estimation were employed.

Results: The successfully measured parameters were comparable with published results (mean diameter (D); 21.1 μm (range 5.8–58 μm), mean axial velocity (Va); 0.50 mm/s (0.11–1 mm/s), blood flow rate (Q); 145.61 pl/s (7.05–1178.81 pl/s) and mean wall shear rate (WSR); 157.31 s⁻¹ (37.37–841.66 s⁻¹)). The method was found to be repeatable. We found the mean difference for D = 0.02 ± 0.31 μm, Va = 0.002 ± 0.02 mm/s, Q = 0.08 ± 11.31 pl/s and WSR = 0.98 ± 8.39 s⁻¹.

Conclusion: The microvasculature can be non-invasively assessed, and haemodynamics quantified using the smartphone-based application described. Future app updates aim to include fully automatic video segment selection and processing.

2.28. Non-Invasive Evaluation of Conjunctival Microvascular Physiology in Patients with Invasive Evidence of Coronary Microvascular Dysfunction

• Julie S Moore ^1,2, Jonathan A Mailey ³, Min Jing ⁴, Agnes Awuah ¹, Paul F Brennan ³, Louise J Robertson ^1,2, M Andrew Nesbit ^1,2, James AD McLaughlin ^2,4, Mark S Spence ³ and Tara Moore ^1,2

¹ 

School of Biomedical Sciences, Ulster University, UK

² 

Integrated Diagnostics Laboratory, Ulster University, UK

³ 

Royal Victoria Hospital, Belfast Health and Social Care Trust, UK

⁴ 

Nanotechnology and Integrated BioEngineering Centre, Ulster University, UK

Introduction: Coronary microvascular disease (CMD) can occur both in isolation, and in combination with epicardial atherosclerotic disease. It is associated with an adverse cardiovascular (CV) prognosis. We hypothesized a possible association between CMD and systemic microvascular dysfunction.

Methods: Conjunctival microvascular haemodynamics were assessed in patients with CMD and compared to age- and sex-matched controls. All participants underwent invasive coronary angiography and microvascular function testing. The CMD cohort (n = 43) was defined by an index of microcirculatory resistance (IMR) ≥ 25 or coronary flow reserve (CFR) < 2. The control cohort (n = 68) had an IMR < 25 and CFR ≥ 2. All patients had no obstructive epicardial coronary arteries. Conjunctival imaging was performed using a slit-lamp coupled with a smartphone. A custom-built application was used to evaluate the conjunctival parameters of vessel diameter (D), axial velocity (Va), cross-sectional velocity (Vs), blood flow rate (Q) and wall shear rate (WSR).

Results: The CMD cohort had reduced Va (0.53 ± 0.04 mm/s vs. 0.55 ± 0.06 mm/s, p = 0.036) and vs. (0.37 ± 0.03 mm/s vs. 0.38 ± 0.04 mm/s, p = 0.038) compared to controls. D was similar between groups (CMD = 24.32 ± 3.18 µm vs. controls = 24.55 ± 3.21 µm, p = 0.59). Numerical reductions were observed in both Q (186 ± 44 pl/s vs. 196 ± 50 pl/s, p = 0.287) and WSR (141 ± 27 s⁻¹ vs. 143 ± 30 s⁻¹, p = 0.624) in the CMD cohort, but differences were not significant.

Conclusion: These findings suggest CMD is associated with alterations in conjunctival microvascular function, that can be identified using this conjunctival microvascular imaging tool and analysis application. This imaging modality may have potential to non-invasively diagnose microvascular dysfunction and hence have clinical utility in CV risk screening.

2.29. Structural, Functional and Molecular Analysis of the Heart in Mouse Models of Spinal Muscular Atrophy

• Nithya N Nair ¹, Rachel A Kline ², Gillian A Gray ³, Thomas M Wishart ² and Lyndsay M Murray ¹

¹ 

Centre for Discovery Brain Sciences, University of Edinburgh, UK

² 

The Roslin Institute and Royal (Dick) School of Veterinary Studies, University of Edinburgh, UK

³ 

Centre for Cardiovascular Science, University of Edinburgh, UK

In spinal muscular atrophy, defects in non-neuronal tissues like the heart could become prominent with new treatments being frequently targeted to the central nervous system. Here, we have performed a thorough study of the structural, functional and molecular basis of the heart in mouse models to explore the underlying basis of cardiac pathology in this disease. An initial analysis of the Smn2B/- mice using published methods revealed structural and functional defects consistent with the existing literature. Many of these defects are no longer significant when normalised to tibia length in order to account for a small body size. However, we do see very subtle changes in certain cardiac parameters which may have to be further investigated. Although an intermediate model of SMA, the hearts of the Smn2B/- mice reveal various dysregulated proteins and pathways from an early pre-symptomatic timepoint. A multi-model analysis also reveals several protein groups and pathways ubiquitously changed in both severe and intermediate mouse models since the day of birth. The functional clustering of both the groups revealed a consistent dysregulation of proteins associated with histone H4, mitochondria and diabetic cardiomyopathy. These changes observed in the intermediate models may underlie and precede the more significant cardiac defects described in the more severe mouse models of SMA. The identification of ubiquitously changed proteins in multiple mouse models since the day of birth indicates the potential to understand the tissue-specific function of Smn in the heart. All these can be further exploited to develop therapeutic strategies to prevent/reverse the heart defects.

2.30. An Important Role for Hypomethylated Integrin Beta-like 1 in Ischaemic Cardiac Fibroblasts

• Lauren Kerrigan ¹, Adam Russell-Hallinan ¹, Kevin Edgar ¹, Carlos Galan-Arriola ², Eduardo Oliver ², Borja Ibanez ², Pat Collier ³, Christine Moreavec ³, Mark Ledwidge ⁴, Ken McDonald ⁵, Sudipto Das ⁵, David Grieve ¹ and Chris Watson ¹

¹ 

Wellcome-Wolfson Institute for Experimental Medicine, Queen’s University Belfast, UK

² 

Centro Nacional de Investigaciones Cardiovasculares (CNIC), Madrid, Spain

³ 

Cleveland Clinic, Ohio, USA

⁴ 

School of Medicine, University College Dublin, Ireland

⁵ 

Royal College of Surgeons Dublin, Ireland

Cardiomyopathy describes cardiac cell alterations which cause structural and functional myocardial abnormalities resulting in heart failure. Ischaemic cardiomyopathy (ICM) is the most common cause of heart failure in the developed world. Myocardial ischaemia causes propagated fibrosis which diminishes left ventricular function. Aberrant epigenetic regulation in cardiac fibroblasts contributes to the development of ICM. DNA methylation occurs when a methyl group is added to a cytosine within a CpG dinucleotide site, often causing gene repression. Abnormal DNA methylation plays a role in the cardiac remodelling in ICM. The functional impact of dysregulated DNA methylation in specific genes in ICM has not been fully elucidated. In this study, we aimed to identify DNA methylation sensitive genes in the human ICM left ventricular (LV) tissue through the integrated use of targeted bisulphite sequencing and RNA sequencing. We identified integrin beta-like 1 (ITGBL1) as a gene of interest though comparative analysis. ITGBL1 was hypomethylated in ICM LV tissue and its gene expression was upregulated. Aberrant expression was validated in both an ischaemic pig model and a mouse model. We examined the effect of ITGBL1 siRNA knockdown on cardiac fibroblast migration to uncover its role in fibroblast activation. Lastly, we assessed DNA methylation regulation as a potential mechanism driving ITGBL1 transcription in ICM by measuring CpG methylation status of specific ITGBL1 gene regions in genomic DNA from ICM LV tissue and from stimulated cardiac fibroblasts. Our findings suggest that ITGBL1 could be a key player in the development of cardiac fibrosis in ICM, and that dysregulated gene expression of ITGBL1 is caused by gene specific hypomethylation.

2.31. Regional Variations in Radiation-Induced Cardiac Toxicity

• Karl Butterworth ¹, Chris Watson ², David Grieve ², Refik Kuburas ¹, Mihaela Ghita ¹ and Gerard Walls ¹

¹ 

The Patrick G Johnston Centre for Cancer Research, UK

² 

Wellcome-Wolfson Institute for Experimental Medicine; Queen’s University Belfast, UK

The heart is one of the most critical dose-limiting organs in patients receiving radiotherapy for thoracic indications. Clinical studies have demonstrated regional variations in the radiosensitivity of heart with the base being a differentially radiosensitive region. In this study, we aimed to apply a translationally relevant mouse model of regional radiosensitivity to characterise the late occurring changes in cardiac function, electrophysiology and gene expression using spatial transcriptomics. Aged mice were irradiated with 16 Gy delivered to the cranial third of the heart using a small animal radiotherapy research platform (SARRP). Echo- and electrocardiography were performed and tissues were collected at 30 weeks for spatial transcriptomics analysis. Base irradiation showed time-dependent loss of cardiac function that did not correlate with mean heart dose (MHD) or the volume of the heart receiving 5 Gy (V5, R² < 0.1). Regional analysis identified 131 genes in the atria and 28 genes in the ventricular region that were differentially expressed compared to unirradiated tissue. This study provides the first evidence of spatially resolved gene expression changes in irradiated tissues. The examination of the regional radiation responses in the heart can further our understanding of radiosensitivity in the cardiac base towards identifying actionable targets for pharmacological intervention or improving cardiac dose constraints.

2.32. Mitigative Potential of Statin Therapy in Base-Mediated Radiation Cardiotoxicity

• Gerard Walls ¹, Mihaela Ghita ¹, Refik Kuburas ¹, Kevin Edgar ², Chris Watson ², David Grieve ², Aidan Cole ¹, Suneil Jain ¹ and Karl Butterworth ¹

¹ 

The Patrick G Johnston Centre for Cancer Research, UK

² 

Wellcome-Wolfson Institute for Experimental Medicine; Queen’s University Belfast, UK

Introduction: Despite modern advances in radiotherapy technology, cardiotoxicity is common among patients with lung, breast and oesophageal cancer, including infarction and arrhythmia. Dose and/or volume dependence for radiation heart disease is lacking but recently the dose received by the base region has been correlated with worse survival. Statins have been shown to reduce the short-term functional effects of whole-heart irradiation in murine models, most likely owing to their pleiotropic antioxidant and anti-inflammatory properties. In this study, we assessed if atorvastatin can mitigate late radiation effects following a clinically relevant partial-heart radiation exposure.

Methods: Female C57BL/6 mice (n = 58) received 16 Gy/1# to the cranial third of the heart using image-guided radiotherapy, or atorvastatin therapy alone, or both. The animals were followed up for 50 weeks with serial echocardiography and electrocardiography every 10 weeks, and blood, heart and lung collections at 10, 30 and 50 weeks.

Results: The LV function measurements up to 30 weeks were collected and analysed to date. There is a statistically significant reduction in the left ventricular ejection fraction (~5%) at 30 weeks in the radiotherapy alone group which is not present in the radiotherapy + atorvastatin group. ECG analysis and serum natriuretic peptides analysis is underway. Histological analysis will include assessment of cardiomyocyte morphology, leucocyte infiltration, microvascular density and fibrosis.

Conclusions: Provisional analyses indicate that atorvastatin protects against left ventricular dysfunction following cardiac base irradiation. If confirmed in the complete analysis, these data would support a clinical trial of statin therapy in patients receiving thoracic radiotherapy.

2.33. Cell Type-Specific Divergence in Gene Expression between Human and Mouse Heart Aids Interpretation of Differential Drug Responses

• Oisín Cappa and David Simpson

• Wellcome-Wolfson Institute for Experimental Medicine, Queen’s University Belfast, UK

Mice are the most commonly used animal model to investigate human biology, being the subject of hundreds of thousands of recent publications, including tens of thousands in cardiovascular research. However, up to 90% of drugs that pass pre-clinical trials in mice fail in human trials due to differences between human and mouse physiology. An ability to predict the physiological differences responsible for these failures would be very advantageous. The development of single-cell RNA-Sequencing has enabled the characterisation of tissues and their constituent cell types at the molecular level. Focusing on the heart, our aim is to characterize differences in gene expression between mice and humans in each cardiac cell type. This would enable the prediction of differences in behaviour of drugs known to target genes differentially expressed between the species. Publicly available human and mouse single-cell RNA-sequencing datasets were identified. By restricting analyses to orthologous genes, human and mouse cells were clustered in a combined analysis into cognate cell types according to shared gene expression profiles using the Seurat package. Differential expression between human and mouse heart was detected in all cell types, with the greatest differences in fibroblasts. Genes previously implicated in cardiovascular disease were present amongst the most species-divergent genes, along with many hundreds of known drug-target genes. The differentially expressed genes in each cardiac cell type will be made publicly available to aid planning of future and interpretation of existing mouse studies. Critically, this information will help explain or predict discrepancies between drug responses observed in mice and humans.

2.34. Stratifying Risk of Acute Kidney Injury in Pre and Post Cardiac Surgery Patients Using a Novel Biomarker-Based Algorithm and Clinical Risk Score

• William T McBride ^1,†, Mary Jo Kurth ^2,†, Gavin McLean ¹, Anna Domanska ², John V Lamont ², Daniel Maguire ², Joanne Watt ², SP FitzGerald ², Ian Young ³, Jijin Joseph ¹ and Mark W Ruddock ²

¹ 

Department of Cardiac Anaesthesia, Belfast Health and Social Care Trust, UK

² 

Randox Laboratories Ltd., Clinical Studies Group, Crumlin, UK

³ 

School of Medicine, Dentistry and Biomedical Sciences, Queen’s University of Belfast, UK

^† 

These authors contributed equally.

Acute kidney injury (AKI) following cardiac surgery significantly increases morbidity and mortality risks. Improving existing clinical methods of identifying patients at risk of perioperative AKI may advance management and treatment options. This study investigated whether a combination of biomarkers and clinical factors pre- and post-cardiac surgery could stratify patients at risk of developing AKI. Patients (n = 401) consecutively scheduled for elective cardiac surgery were prospectively studied. Clinical data were recorded and blood samples were tested for 31 biomarkers. Areas under receiver operating characteristic (AUROCs) were generated for biomarkers pre- and post-operatively to stratify patients at risk of AKI. Pre-operatively, sTNFR1 had the highest predictive ability to identify risk of developing AKI post-operatively (AUROC 0.748). Post-operatively, a combination of H-FABP, midkine and sTNFR2 had the highest predictive ability to identify AKI risk (AUROC 0.836). Pre-operative clinical risk factors included patient age, body mass index and diabetes. Peri-operative factors included cardio pulmonary bypass, cross-clamp and operation times, intra-aortic balloon pump, blood products and resternotomy. Combining biomarker risk score (BRS) with clinical risk score (CRS) enabled pre- and post-operative assignment of patients to AKI risk categories. Combining BRS with CRS will allow the better management of cardiac patients at risk of developing AKI.