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Cell therapy is a potential novel treatment for cardiac regeneration and numerous studies have attempted to transplant cells to regenerate the myocardium lost during myocardial infarction. To date, only minimal improvements to cardiac function have been reported. This is likely to be the result of low cell retention and survival following transplantation. This study aimed to improve the delivery and engraftment of viable cells by using an injectable microcarrier that provides an implantable, biodegradable substrate for attachment and growth of cardiomyocytes derived from induced pluripotent stem cells (iPSC). We describe the fabrication and characterisation of Thermally Induced Phase Separation (TIPS) microcarriers and their surface modification to enable iPSC-derived cardiomyocyte attachment in xeno-free conditions is described. The selected formulation resulted in iPSC attachment, expansion, and retention of pluripotent phenotype. Differentiation of iPSC into cardiomyocytes on the microcarriers is investigated in comparison with culture on 2D tissue culture plastic surfaces. Microcarrier culture is shown to support culture of a mature cardiomyocyte phenotype, be compatible with injectable delivery, and reduce anoikis. The findings from this study demonstrate that TIPS microcarriers provide a supporting matrix for culturing iPSC and iPSC-derived cardiomyocytes in vitro and are suitable as an injectable cell-substrate for cardiac regeneration., (© 2024 The Author(s). Advanced Science published by Wiley‐VCH GmbH.)

Protein-based biomaterial use is expanding within medicine, together with the demand to visualize their placement and behavior in vivo. However, current medical imaging techniques struggle to differentiate between protein-based implants and surrounding tissue. Here a fast, simple, and translational solution for tracking transplanted protein-based scaffolds is presented using X-ray CT-facilitating long-term, non-invasive, and high-resolution imaging. X-ray visible scaffolds are engineered by selectively iodinating tyrosine residues under mild conditions using readily available reagents. To illustrate translatability, a clinically approved hernia repair mesh (based on decellularized porcine dermis) is labeled, preserving morphological and mechanical properties. In a mouse model of mesh implantation, implants retain marked X-ray contrast up to 3 months, together with an unchanged degradation rate and inflammatory response. The technique's compatibility is demonstrated with a range of therapeutically relevant protein formats including bovine, porcine, and jellyfish collagen, as well as silk sutures, enabling a wide range of surgical and regenerative medicine uses. This solution tackles the challenge of visualizing implanted protein-based biomaterials, which conventional imaging methods fail to differentiate from endogenous tissue. This will address previously unanswered questions regarding the accuracy of implantation, degradation rate, migration, and structural integrity, thereby accelerating optimization and safe translation of therapeutic biomaterials., (© 2023 The Authors. Advanced Science published by Wiley-VCH GmbH.)

Medical therapies achieve their control at expense to the patient in the form of a range of toxicities, which incur costs and diminish quality of life. Magnetic resonance navigation is an emergent technique that enables image-guided remote-control of magnetically labeled therapies and devices in the body, using a magnetic resonance imaging (MRI) system. Minimally INvasive IMage-guided Ablation (MINIMA), a novel, minimally invasive, MRI-guided ablation technique, which has the potential to avoid traditional toxicities, is presented. It comprises a thermoseed navigated to a target site using magnetic propulsion gradients generated by an MRI scanner, before inducing localized cell death using an MR-compatible thermoablative device. The authors demonstrate precise thermoseed imaging and navigation through brain tissue using an MRI system (0.3 mm), and they perform thermoablation in vitro and in vivo within subcutaneous tumors, with the focal ablation volume finely controlled by heating duration. MINIMA is a novel theranostic platform, combining imaging, navigation, and heating to deliver diagnosis and therapy in a single device., (© 2022 The Authors. Advanced Science published by Wiley-VCH GmbH.)

Cell-based therapies have been making great advances toward clinical reality. Despite the increase in trial activity, few therapies have successfully navigated late-phase clinical trials and received market authorization. One possible explanation for this is that additional tools and technologies to enable their development have only recently become available. To support the safety evaluation of cell therapies, the Health and Environmental Sciences Institute Cell Therapy-Tracking, Circulation and Safety Committee, a multisector collaborative committee, polled the attendees of the 2017 International Society for Cell & Gene Therapy conference in London, UK, to understand the gaps and needs that cell therapy developers have encountered regarding safety evaluations in vivo. The goal of the survey was to collect information to inform stakeholders of areas of interest that can help ensure the safe use of cellular therapeutics in the clinic. This review is a response to the cellular imaging interests of those respondents. The authors offer a brief overview of available technologies and then highlight the areas of interest from the survey by describing how imaging technologies can meet those needs. The areas of interest include imaging of cells over time, sensitivity of imaging modalities, ability to quantify cells, imaging cellular survival and differentiation and safety concerns around adding imaging agents to cellular therapy protocols. The Health and Environmental Sciences Institute Cell Therapy-Tracking, Circulation and Safety Committee believes that the ability to understand therapeutic cell fate is vital for determining and understanding cell therapy efficacy and safety and offers this review to aid in those needs. An aim of this article is to share the available imaging technologies with the cell therapy community to demonstrate how these technologies can accomplish unmet needs throughout the translational process and strengthen the understanding of cellular therapeutics., (Copyright © 2021 International Society for Cell & Gene Therapy. Published by Elsevier Inc. All rights reserved.)

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

Early measurements of tissue viability after myocardial infarction (MI) are essential for accurate diagnosis and treatment planning but are challenging to obtain. Here, manganese, a calcium analogue and clinically approved magnetic resonance imaging (MRI) contrast agent, is used as an imaging biomarker of myocardial viability in the first hours after experimental MI. Safe Mn 2+ dosing is confirmed by measuring in vitro beating rates, calcium transients, and action potentials in cardiomyocytes, and in vivo heart rates and cardiac contractility in mice. Quantitative T1 mapping-manganese-enhanced MRI (MEMRI) reveals elevated and increasing Mn 2+ uptake in viable myocardium remote from the infarct, suggesting MEMRI offers a quantitative biomarker of cardiac inotropy. MEMRI evaluation of infarct size at 1 h, 1 and 14 days after MI quantifies myocardial viability earlier than the current gold-standard technique, late-gadolinium-enhanced MRI. These data, coupled with the re-emergence of clinical Mn 2+ -based contrast agents open the possibility of using MEMRI for direct evaluation of myocardial viability early after ischemic onset in patients., (© 2021 The Authors. Advanced Science published by Wiley-VCH GmbH.)

Interleukin (IL)-6 is an emerging therapeutic target in myocardial infarction (MI). IL-6 has 2 distinct signaling pathways: trans-signaling, which mediates inflammation, and classic signaling, which also has anti-inflammatory effects. The novel recombinant fusion protein sgp130Fc achieves exclusive trans-signaling blockade, whereas anti-IL-6 antibodies (Abs) result in panantagonism. In a rat model of reperfused MI, sgp130Fc, but not anti-IL-6-Ab, attenuated neutrophil and macrophage infiltration into the myocardium, reduced infarct size, and preserved cardiac function 28 days after MI. These data demonstrate the efficacy of exclusive IL-6 trans-signaling blockade and support further investigation of sgp130Fc as a potential novel therapy in MI., Competing Interests: This work was supported by the Wellcome Trust (108735/Z/15/Z to Dr. George and 212937/Z/18/Z to Dr. Stuckey), the British Heart Foundation (FS/15/33/31608 and RM/17/1/33377 to Dr. Stuckey), the Medical Research Council (MR/R026416/1 to Dr. Stuckey), the National Institute for Health Research (Senior Investigator: Dr, Hingorani), and a King Scholarship of Malaysia (Ms. Jasmin). Dr. Woollard is an employee of AstraZeneca. No funding or support was received from AstraZeneca. All other authors have reported that they have no relationships relevant to the contents of this paper to disclose., (© 2021 The Authors.)

Significance: Tumor detection and margin delineation are essential for successful tumor resection. However, postsurgical positive margin rates remain high for many cancers. Raman spectroscopy has shown promise as a highly accurate clinical spectroscopic diagnostic modality, but its margin delineation capabilities are severely limited by the need for pointwise application., Aim: We aim to extend Raman spectroscopic diagnostics and develop a multimodal computer vision-based diagnostic system capable of both the detection and identification of suspicious lesions and the precise delineation of disease margins., Approach: We first apply visual tracking of a Raman spectroscopic probe to achieve real-time tumor margin delineation. We then combine this system with protoporphyrin IX fluorescence imaging to achieve fluorescence-guided Raman spectroscopic margin delineation., Results: Our system enables real-time Raman spectroscopic tumor margin delineation for both ex vivo human tumor biopsies and an in vivo tumor xenograft mouse model. We then further demonstrate that the addition of protoporphyrin IX fluorescence imaging enables fluorescence-guided Raman spectroscopic margin delineation in a tissue phantom model., Conclusions: Our image-guided Raman spectroscopic probe-tracking system enables tumor margin delineation and is compatible with both white light and fluorescence image guidance, demonstrating the potential for our system to be developed toward clinical tumor resection surgeries.

Theranostics, the combination of diagnosis and therapy, has long held promise as a means to achieving personalised precision cancer treatments. However, despite its potential, theranostics has yet to realise significant clinical translation, largely due the complexity and overriding toxicity concerns of existing theranostic nanoparticle strategies. Methods: Here, we present an alternative nanoparticle-free theranostic approach based on simultaneous Raman spectroscopy and photodynamic therapy (PDT) in an integrated clinical platform for cancer theranostics. Results: We detail the compatibility of Raman spectroscopy and PDT for cancer theranostics, whereby Raman spectroscopic diagnosis can be performed on PDT photosensitiser-positive cells and tissues without inadvertent photosensitiser activation/photobleaching or impaired diagnostic capacity. We further demonstrate that our theranostic platform enables in vivo tumour diagnosis, treatment, and post-treatment molecular monitoring in real-time. Conclusion: This system thus achieves effective theranostic performance, providing a promising new avenue towards the clinical realisation of theranostics., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

Key Points: Maternal hypertension is associated with increased rates of pregnancy pathologies, including fetal growth restriction, due at least in part to reductions in nitric oxide (NO) bioavailability and associated vascular dysfunction. Dietary nitrate supplementation, from beetroot juice (BRJ), has been shown to increase NO bioavailability and improve cardiovascular function in both preclinical and clinical studies. This study is the first to investigate effects of dietary nitrate supplementation in a pregnant animal model. Importantly, the effects of nitrate-containing BRJ were compared with both 'placebo' (nitrate-depleted) BRJ as well as water to control for potential nitrate-independent effects. Our data show novel, nitrate-independent effects of BRJ to lower blood pressure and improve vascular function in endothelial nitric oxide synthase knockout (eNOS -/- ) mice. These findings suggest potential beneficial effects of BRJ supplementation in pregnancy, and emphasize the importance of accounting for nitrate-independent effects of BRJ in study design and interpretation., Abstract: Maternal hypertension is associated with adverse pregnancy outcomes, including fetal growth restriction (FGR), due in part to reductions in nitric oxide (NO) bioavailability. We hypothesized that maternal dietary nitrate administration would increase NO bioavailability to reduce systolic blood pressure (SBP), improve vascular function and increase fetal growth in pregnant endothelial NO synthase knockout (eNOS -/- ) mice, which exhibit hypertension, endothelial dysfunction and FGR. Pregnant wildtype (WT) and eNOS -/- mice were supplemented with nitrate-containing beetroot juice (BRJ+) from gestational day (GD) 12.5. Control mice received an equivalent dose of nitrate-depleted BRJ (BRJ-) or normal drinking water. At GD17.5, maternal SBP was measured; at GD18.5, maternal nitrate/nitrite concentrations, uterine artery (UtA) blood flow and endothelial function were assessed, and pregnancy outcomes were determined. Plasma nitrate concentrations were increased in both WT and eNOS -/- mice supplemented with BRJ+ (P < 0.001), whereas nitrite concentrations were increased only in eNOS -/- mice (P < 0.001). BRJ- did not alter nitrate/nitrite concentrations. SBP was lowered and UtA endothelial function was enhanced in eNOS -/- mice supplemented with either BRJ+ or BRJ-, indicating nitrate-independent effects of BRJ. Improvements in endothelial function in eNOS -/- mice were abrogated in the presence of 25 mm KCl, implicating enhanced EDH signalling in BRJ- treated animals. At GD18.5, eNOS -/- fetuses were significantly smaller than WT animals (P < 0.001), but BRJ supplementation did not affect fetal weight. BRJ may be a beneficial intervention in pregnancies associated with hypertension, endothelial dysfunction and reduced NO bioavailability. Our data showing biological effects of non-nitrate components of BRJ have implications for both interpretation of previous findings and in the design of future clinical trials., (© 2020 The Authors. The Journal of Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society.)

Alginate hydrogels are cross-linked polymers with high water content, tuneable chemical and material properties, and a range of biomedical applications including drug delivery, tissue engineering, and cell therapy. However, their similarity to soft tissue often renders them undetectable within the body using conventional bio-medical imaging techniques. This leaves much unknown about their behaviour in vivo, posing a challenge to therapy development and validation. To address this, we report a novel, fast, and simple method of incorporating the nuclear imaging radio-metal 111 In into the structure of alginate hydrogels by utilising its previously-undescribed capacity as an ionic cross-linking agent. This enabled non-invasive in vivo nuclear imaging of hydrogel delivery and retention across the whole body, over time, and across a range of model therapies including: nasal and oral drug delivery, stem cell transplantation, and cardiac tissue engineering. This information will facilitate the development of novel therapeutic hydrogel formulations, encompassing alginate, across disease categories., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed., (Copyright © 2020 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Cell-based therapies are promising treatments for various kidney diseases. However, the major hurdle in initiating therapeutic responses is the inefficiency of injection routes to deliver cells to the kidney parenchyma. Systemic injection, such as intravenous injection only delivers a small proportion of cells to the kidney. Whereas direct delivery, such as renal artery injection requires surgical procedures. A minimally invasive renal artery injection was therefore developed to enhance cell delivery to kidney. In this study, luciferase expressing human adipocyte derived stem cells (ADSC) were labelled with gold nanorods (GNR) and injected into the renal artery using ultrasound guidance. The ADSCs were tracked using bioluminescence and photoacoustic imaging serially over 7 days. Imaging confirmed that the majority of signal was within the kidney, indicative of successful injection and that the cells remained viable for 3 days. Histology showed co-localization of GNRs with ADSC staining throughout the kidney with no indication of injury caused by injection. These findings demonstrate that ultrasound-guided renal artery injection is feasible in mice and can successfully deliver a large proportion of cells which are retained within the kidney for 3 days. Therefore, the techniques developed here will be useful for optimising cell therapy in kidney diseases.

Optical imaging in clinical and preclinical settings can provide a wealth of biological information, particularly when coupled with targetted nanoparticles, but optical scattering and absorption limit the depth and resolution in both animal and human subjects. Two new hybrid approaches are presented, using the penetrating power of X-rays to increase the depth of optical imaging. Foremost, we demonstrate the excitation by X-rays of quantum-dots (QD) emitting in the near-infrared (NIR), using a clinical X-ray system to map the distribution of QDs at depth in whole mouse. We elicit a clear, spatially-resolved NIR signal from deep organs (brain, liver and kidney) with short (1 second) exposures and tolerable radiation doses that will permit future in vivo applications. Furthermore, X-ray-excited endogenous emission is also detected from whole mouse. The use of keV X-rays to excite emission from QDs and tissue represent novel biomedical imaging technologies, and exploit emerging QDs as optical probes for spatial-temporal molecular imaging at greater depth than previously possible.

Background: Transarterial chemoembolization (TACE) is the current standard of care for patients with intermediate-stage hepatocellular carcinoma (HCC) and is also a treatment option for patients with liver metastases from colorectal cancer. However, TACE is not a curative treatment, and tumor progression occurs in more than half of the patients treated. Despite advances and technical refinements of TACE, including the introduction of drug-eluting beads-TACE, the clinical efficacy of TACE has not been optimized, and improved arterial therapies are required., Objective: The primary objectives of the VEROnA study are to evaluate the safety and tolerability of vandetanib-eluting radiopaque embolic beads (BTG-002814) in patients with resectable liver malignancies and to determine concentrations of vandetanib and the N-desmethyl metabolite in plasma and resected liver following treatment with BTG-002814., Methods: The VEROnA study is a first-in-human, open-label, single-arm, phase 0, window-of-opportunity study of BTG-002814 (containing 100 mg vandetanib) delivered transarterially, 7 to 21 days before surgery in patients with resectable liver malignancies. Eligible patients have a diagnosis of colorectal liver metastases, or HCC (Childs Pugh A), diagnosed histologically or radiologically, and are candidates for liver surgery. All patients are followed up for 28 days following surgery. Secondary objectives of this study are to evaluate the anatomical distribution of BTG-002814 on noncontrast-enhanced imaging, to evaluate histopathological features in the surgical specimen, and to assess changes in blood flow on dynamic contrast-enhanced magnetic resonance imaging following treatment with BTG-002814. Exploratory objectives of this study are to study blood biomarkers with the potential to identify patients likely to respond to treatment and to correlate the distribution of BTG-002814 on imaging with pathology by 3-dimensional modeling., Results: Enrollment for the study was completed in February 2019. Results of a planned interim analysis were reviewed by a safety committee after the first 3 patients completed follow-up. The recommendation of the committee was to continue the study without any changes to the dose or trial design, as there were no significant unexpected toxicities related to BTG-002814., Conclusions: The VEROnA study is studying the feasibility of administering BTG-002814 to optimize the use of this novel technology as liver-directed therapy for patients with primary and secondary liver cancer., Trial Registration: ClinicalTrial.gov NCT03291379; https://clinicaltrials.gov/ct2/show/NCT03291379., International Registered Report Identifier (irrid): DERR1-10.2196/13696., (©Laura Beaton, Henry F J Tregidgo, Sami A Znati, Sharon Forsyth, Matthew J Clarkson, Steven Bandula, Manil Chouhan, Helen L Lowe, May Zaw Thin, Julian Hague, Dinesh Sharma, Joerg-Matthias Pollok, Brian R Davidson, Jowad Raja, Graham Munneke, Daniel J Stuckey, Zainab A Bascal, Paul E Wilde, Sarah Cooper, Samantha Ryan, Peter Czuczman, Eveline Boucher, John A Hartley, Andrew L Lewis, Marnix Jansen, Tim Meyer, Ricky A Sharma. Originally published in JMIR Research Protocols (http://www.researchprotocols.org), 02.10.2019.)

Adult hearts respond to increased workload such as prolonged stress or injury, by undergoing hypertrophic growth. During this process, the early adaptive responses are important for maintaining cardiac output whereas at later stages, pathological responses such as cardiomyocyte apoptosis and fibrosis cause adverse remodelling, that can progress to heart failure. Yet the factors that control transition from adaptive responses to pathological remodelling in the heart are not well understood. Here we describe the POU4F2/Brn-3b transcription factor (TF) as a novel regulator of adaptive hypertrophic responses in adult hearts since Brn-3b mRNA and protein are increased in angiotensin-II (AngII) treated mouse hearts with concomitant hypertrophic changes [increased heart weight:body weight (HW:BW) ratio]. These effects occur specifically in cardiomyocytes because Brn-3b expression is increased in AngII-treated primary cultures of neonatal rat ventricular myocytes (NRVM) or foetal heart-derived H9c2 cells, which undergo characteristic sarcomeric re-organisation seen in hypertrophic myocytes and express hypertrophic markers, ANP/βMHC. The Brn-3b promoter is activated by known hypertrophic signalling pathways e.g. p42/p44 mitogen-activated protein kinase (MAPK/ERK1/2) or calcineurin (via NFAT). Brn-3b target genes, e.g. cyclin D1, GLUT4 and Bax, are increased at different stages following AngII treatment, supporting distinct roles in cardiac responses to stress. Furthermore, hearts from male Brn-3b KO mutant mice display contractile dysfunction at baseline but also attenuated hypertrophic responses to AngII treatment. Hearts from AngII-treated male Brn-3b KO mice develop further contractile dysfunction linked to extensive fibrosis/remodelling. Moreover, known Brn-3b target genes, e.g. GLUT4, are reduced in AngII-treated Brn-3b KO hearts, suggesting that Brn-3b and its target genes are important in driving adaptive hypertrophic responses in stressed heart.

In utero gene therapy (IUGT) to the fetal hematopoietic compartment could be used to treat congenital blood disorders such as β-thalassemia. A humanised mouse model of β-thalassemia was used, in which heterozygous animals are anaemic with splenomegaly and extramedullary hematopoiesis. Intrahepatic in utero injections of a β globin-expressing lentiviral vector (GLOBE), were performed in fetuses at E13.5 of gestation. We analysed animals at 12 and 32 weeks of age, for vector copy number in bone marrow, peripheral blood liver and spleen and we performed integration site analysis. Compared to noninjected heterozygous animals IUGT normalised blood haemoglobin levels and spleen weight. Integration site analysis showed polyclonality. The left ventricular ejection fraction measured using magnetic resonance imaging (MRI) in treated heterozygous animals was similar to that of normal non-β-thalassemic mice but significantly higher than untreated heterozygous thalassemia mice suggesting that IUGT ameliorated poor cardiac function. GLOBE LV-mediated IUGT normalised the haematological and anatomical phenotype in a heterozygous humanised model of β-thalassemia.

Heart disease is a paramount cause of global death and disability. Although cardiomyocyte death plays a causal role and its suppression would be logical, no clinical counter-measures target the responsible intracellular pathways. Therapeutic progress has been hampered by lack of preclinical human validation. Mitogen-activated protein kinase kinase kinase kinase-4 (MAP4K4) is activated in failing human hearts and relevant rodent models. Using human induced-pluripotent-stem-cell-derived cardiomyocytes (hiPSC-CMs) and MAP4K4 gene silencing, we demonstrate that death induced by oxidative stress requires MAP4K4. Consequently, we devised a small-molecule inhibitor, DMX-5804, that rescues cell survival, mitochondrial function, and calcium cycling in hiPSC-CMs. As proof of principle that drug discovery in hiPSC-CMs may predict efficacy in vivo, DMX-5804 reduces ischemia-reperfusion injury in mice by more than 50%. We implicate MAP4K4 as a well-posed target toward suppressing human cardiac cell death and highlight the utility of hiPSC-CMs in drug discovery to enhance cardiomyocyte survival., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

The homeodomain transcription factor NKX2-5 is known to be essential for both normal heart development and for heart function. But little is yet known about the identities of its downstream effectors or their function during differentiation of cardiac progenitor cells (CPCs). We have used transgenic analysis and CRISPR-mediated ablation to identify a cardiac enhancer of the Furin gene. The Furin gene, encoding a proprotein convertase, is directly repressed by NKX2-5. Deletion of Furin in CPCs is embryonic lethal, with mutant hearts showing a range of abnormalities in the outflow tract. Those defects are associated with a reduction in proliferation and premature differentiation of the CPCs. Deletion of Furin in differentiated cardiomyocytes results in viable adult mutant mice showing an elongation of the PR interval, a phenotype that is consistent with the phenotype of mice and human mutant for Nkx2-5. Our results show that Furin mediate some aspects of Nkx2-5 function in the heart., Competing Interests: The authors have declared that no competing interests exist.

Background: In the era of increasingly successful corrective interventions in patients with congenital heart disease (CHD), global and regional myocardial remodeling are emerging as important sources of long-term morbidity/mortality. Changes in organization of the myocardium in CHD, and in its mechanical properties, conduction, and blood supply, result in altered myocardial function both before and after surgery. To gain a better understanding and develop appropriate and individualized treatment strategies, the microscopic organization of cardiomyocytes, and their integration at a macroscopic level, needs to be completely understood. The aim of this study is to describe, for the first time, in 3 dimensions and nondestructively the detailed remodeling of cardiac microstructure present in a human fetal heart with complex CHD., Methods and Results: Synchrotron X-ray phase-contrast imaging was used to image an archival midgestation formalin-fixed fetal heart with right isomerism and complex CHD and compare with a control fetal heart. Analysis of myocyte aggregates, at detail not accessible with other techniques, was performed. Macroanatomic and conduction system changes specific to the disease were clearly observable, together with disordered myocyte organization in the morphologically right ventricle myocardium. Electrical activation simulations suggested altered synchronicity of the morphologically right ventricle., Conclusions: We have shown the potential of X-ray phase-contrast imaging for studying cardiac microstructure in the developing human fetal heart at high resolution providing novel insight while preserving valuable archival material for future study. This is the first study to show myocardial alterations occur in complex CHD as early as midgestation.

Cardiac fibrosis is implicit in all forms of heart disease but there are no effective treatments. In this report, we investigate the role of the multi-functional enzyme Transglutaminase 2 (TG2) in cardiac fibrosis and assess its potential as a therapeutic target. Here we describe the use a highly selective TG2 small-molecule inhibitor to test the efficacy of TG2 inhibition as an anti-fibrotic therapy for heart failure employing two different in vivo models of cardiac fibrosis: Progressively induced interstitial cardiac fibrosis by pressure overload using angiotensin II infusion: Acutely induced focal cardiac fibrosis through myocardial infarction by ligation of the left anterior descending coronary artery (AMI model). In the AMI model, in vivo MRI showed that the TG2 inhibitor 1-155 significantly reduced infarct size by over 50% and reduced post-infarct remodelling at 20 days post insult. In both models, Sirius red staining for collagen deposition and levels of the TG2-mediated protein crosslink ε(γ-glutamyl)lysine were significantly reduced. No cardiac rupture or obvious signs of toxicity were observed. To provide a molecular mechanism for TG2 involvement in cardiac fibrosis, we show that both TGFβ1-induced transition of cardiofibroblasts into myofibroblast-like cells and TGFβ1-induced EndMT, together with matrix deposition, can be attenuated by the TG2 selective inhibitor 1-155, suggesting a new role for TG2 in regulating TGFβ1 signalling in addition to its role in latent TGFβ1 activation. In conclusion, TG2 has a role in cardiac fibrosis through activation of myofibroblasts and matrix deposition. TG2 inhibition using a selective small-molecule inhibitor can attenuate cardiac fibrosis.

Retention and survival of transplanted cells are major limitations to the efficacy of regenerative medicine, with short-term paracrine signals being the principal mechanism underlying current cell therapies for heart repair. Consequently, even improvements in short-term durability may have a potential impact on cardiac cell grafting. We have developed a multimodal hydrogel-based platform comprised of a poly(ethylene glycol) network cross-linked with bioactive peptides functionalized with Gd(III) in order to monitor the localization and retention of the hydrogel in vivo by magnetic resonance imaging. In this study, we have tailored the material for cardiac applications through the inclusion of a heparin-binding peptide (HBP) sequence in the cross-linker design and formulated the gel to display mechanical properties resembling those of cardiac tissue. Luciferase-expressing cardiac stem cells (CSC-Luc2) encapsulated within these gels maintained their metabolic activity for up to 14 days in vitro. Encapsulation in the HBP hydrogels improved CSC-Luc2 retention in the mouse myocardium and hind limbs at 3 days by 6.5- and 12- fold, respectively. Thus, this novel heparin-binding based, Gd(III)-tagged hydrogel and CSC-Luc2 platform system demonstrates a tailored, in vivo detectable theranostic cell delivery system that can be implemented to monitor and assess the transplanted material and cell retention.

Systemic autoimmune diseases such as systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA) show significant heart involvement and cardiovascular morbidity, which can be due to systemically increased levels of inflammation or direct autoreactivity targeting cardiac tissue. Despite high clinical relevance, cardiac damage secondary to systemic autoimmunity lacks inducible rodent models. Here, we characterise immune-mediated cardiac tissue damage in a new model of SLE induced by topical application of the Toll-like receptor 7/8 (TLR7/8) agonist Resiquimod. We observe a cardiac phenotype reminiscent of autoimmune-mediated dilated cardiomyopathy, and identify auto-antibodies as major contributors to cardiac tissue damage. Resiquimod-induced heart disease is a highly relevant mouse model for mechanistic and therapeutic studies aiming to protect the heart during autoimmunity., (© 2017. Published by The Company of Biologists Ltd.)

β-thalassemia (βT) is a genetic blood disorder causing profound and life threatening anemia. Current clinical management of βT is a lifelong dependence on regular blood transfusions, a consequence of which is systemic iron overload leading to acute heart failure. Recent developments in gene and chelation therapy give hope of better prognosis for patients, but successful translation to clinical practice is hindered by the lack of thorough preclinical testing using representative animal models and clinically relevant quantitative biomarkers. Here we demonstrate a quantitative and non-invasive preclinical Magnetic Resonance Imaging (MRI) platform for the assessment of βT in the γβ 0 /γβ A humanized mouse model of βT. Changes in the quantitative MRI relaxation times as well as severe splenomegaly were observed in the heart, liver and spleen in βT. These data showed high sensitivity to iron overload and a strong relationship between quantitative MRI relaxation times and hepatic iron content. Importantly these changes preceded the onset of iron overload cardiomyopathy, providing an early biomarker of disease progression. This work demonstrates that multiparametric MRI is a powerful tool for the assessment of preclinical βT, providing sensitive and quantitative monitoring of tissue iron sequestration and cardiac dysfunction- parameters essential for the preclinical development of new therapeutics.

Key Points: The strength, functional significance and origins of parasympathetic innervation of the left ventricle remain controversial. This study tested the hypothesis that parasympathetic control of left ventricular contractility is provided by vagal preganglionic neurones of the dorsal motor nucleus (DVMN). Under β-adrenoceptor blockade combined with spinal cord (C1) transection (to remove sympathetic influences), systemic administration of atropine increased left ventricular contractility in rats anaesthetized with urethane, confirming the existence of a tonic inhibitory muscarinic influence on cardiac inotropy. Increased left ventricular contractility in anaesthetized rats was observed when DVMN neurones were silenced. Functional neuroanatomical mapping revealed that vagal preganglionic neurones that have an impact on left ventricular contractility are located in the caudal region of the left DVMN. These neurones provide functionally significant parasympathetic control of left ventricular inotropy., Abstract: The strength, functional significance and origins of direct parasympathetic innervation of the left ventricle (LV) remain controversial. In the present study we used an anaesthetized rat model to first confirm the presence of tonic inhibitory vagal influence on LV inotropy. Using genetic neuronal targeting and functional neuroanatomical mapping we tested the hypothesis that parasympathetic control of LV contractility is provided by vagal preganglionic neurones located in the dorsal motor nucleus (DVMN). It was found that under systemic β-adrenoceptor blockade (atenolol) combined with spinal cord (C1) transection (to remove sympathetic influences), intravenous administration of atropine increases LV contractility in rats anaesthetized with urethane, but not in animals anaesthetized with pentobarbital. Increased LV contractility in rats anaesthetized with urethane was also observed when DVMN neurones targeted bilaterally to express an inhibitory Drosophila allatostatin receptor were silenced by application of an insect peptide allatostatin. Microinjections of glutamate and muscimol to activate or inhibit neuronal cell bodies in distinct locations along the rostro-caudal extent of the left and right DVMN revealed that vagal preganglionic neurones, which have an impact on LV contractility, are located in the caudal region of the left DVMN. Changes in LV contractility were only observed when this subpopulation of DVMN neurones was activated or inhibited. These data confirm the existence of a tonic inhibitory muscarinic influence on LV contractility. Activity of a subpopulation of DVMN neurones provides functionally significant parasympathetic control of LV contractile function., (© 2016 The Authors. The Journal of Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society.)

Background: Research using orthotopic and transgenic models of cancer requires imaging methods to non-invasively quantify tumour burden. As the choice of appropriate imaging modality is wide-ranging, this study aimed to compare low-field (1T) magnetic resonance imaging (MRI), a novel and relatively low-cost system, against established preclinical techniques: bioluminescence imaging (BLI), ultrasound imaging (US), and high-field (9.4T) MRI., Methods: A model of colorectal metastasis to the liver was established in eight mice, which were imaged with each modality over four weeks post-implantation. Tumour burden was assessed from manually segmented regions., Results: All four imaging systems provided sufficient contrast to detect tumours in all of the mice after two weeks. No significant difference was detected between tumour doubling times estimated by low-field MRI, ultrasound imaging or high-field MRI. A strong correlation was measured between high-field MRI estimates of tumour burden and all the other modalities (p < 0.001, Pearson)., Conclusion: These results suggest that both low-field MRI and ultrasound imaging are accurate modalities for characterising the growth of preclinical tumour models.

Cardiac progenitor/stem cells in adult hearts represent an attractive therapeutic target for heart regeneration, though (inter)-relationships among reported cells remain obscure. Using single-cell qRT-PCR and clonal analyses, here we define four subpopulations of cardiac progenitor/stem cells in adult mouse myocardium all sharing stem cell antigen-1 (Sca1), based on side population (SP) phenotype, PECAM-1 (CD31) and platelet-derived growth factor receptor-α (PDGFRα) expression. SP status predicts clonogenicity and cardiogenic gene expression (Gata4/6, Hand2 and Tbx5/20), properties segregating more specifically to PDGFRα(+) cells. Clonal progeny of single Sca1(+) SP cells show cardiomyocyte, endothelial and smooth muscle lineage potential after cardiac grafting, augmenting cardiac function although durable engraftment is rare. PDGFRα(-) cells are characterized by Kdr/Flk1, Cdh5, CD31 and lack of clonogenicity. PDGFRα(+)/CD31(-) cells derive from cells formerly expressing Mesp1, Nkx2-5, Isl1, Gata5 and Wt1, distinct from PDGFRα(-)/CD31(+) cells (Gata5 low; Flk1 and Tie2 high). Thus, PDGFRα demarcates the clonogenic cardiogenic Sca1(+) stem/progenitor cell.

In Takotsubo cardiomyopathy, the left ventricle shows apical ballooning combined with basal hypercontractility. Both clinical observations in humans and recent experimental work on isolated rat ventricular myocytes suggest the dominant mechanisms of this syndrome are related to acute catecholamine overload. However, relating observed differences in single cells to the capacity of such alterations to result in the extreme changes in ventricular shape seen in Takotsubo syndrome is difficult. By using a computational model of the rat left ventricle, we investigate which mechanisms can give rise to the typical shape of the ventricle observed in this syndrome. Three potential dominant mechanisms related to effects of β-adrenergic stimulation were considered: apical-basal variation of calcium transients due to differences in L-type and sarco(endo)plasmic reticulum Ca(2+)-ATPase activation, apical-basal variation of calcium sensitivity due to differences in troponin I phosphorylation, and apical-basal variation in maximal active tension due to, e.g., the negative inotropic effects of p38 MAPK. Furthermore, we investigated the interaction of these spatial variations in the presence of a failing Frank-Starling mechanism. We conclude that a large portion of the apex needs to be affected by severe changes in calcium regulation or contractile function to result in apical ballooning, and smooth linear variation from apex to base is unlikely to result in the typical ventricular shape observed in this syndrome. A failing Frank-Starling mechanism significantly increases apical ballooning at end systole and may be an important additional factor underpinning Takotsubo syndrome., (Copyright © 2014 the American Physiological Society.)

Background: Myocardial infarction (MI) is one of the leading causes of heart failure. An increasing body of evidence links alterations in cardiac metabolism and mitochondrial function with the progression of heart disease. The aim of this work was to, therefore, follow the in vivo mitochondrial metabolic alterations caused by MI, thereby allowing a greater understanding of the interplay between metabolic and functional abnormalities., Methods and Results: Using hyperpolarized carbon-13 ((13)C)-magnetic resonance spectroscopy, in vivo alterations in mitochondrial metabolism were assessed for 22 weeks after surgically induced MI with reperfusion in female Wister rats. One week after MI, there were no detectable alterations in in vivo cardiac mitochondrial metabolism over the range of ejection fractions observed (from 28% to 84%). At 6 weeks after MI, in vivo mitochondrial Krebs cycle activity was impaired, with decreased (13)C-label flux into citrate, glutamate, and acetylcarnitine, which correlated with the degree of cardiac dysfunction. These changes were independent of alterations in pyruvate dehydrogenase flux. By 22 weeks, alterations were also seen in pyruvate dehydrogenase flux, which decreased at lower ejection fractions. These results were confirmed using in vitro analysis of enzyme activities and metabolomic profiles of key intermediates., Conclusions: The in vivo decrease in Krebs cycle activity in the 6-week post-MI heart may represent an early maladaptive phase in the metabolic alterations after MI in which reductions in Krebs cycle activity precede a reduction in pyruvate dehydrogenase flux. Changes in mitochondrial metabolism in heart disease are progressive and proportional to the degree of cardiac impairment., (© 2014 American Heart Association, Inc.)

Background: Diffuse interstitial fibrosis is present in diverse cardiomyopathies and associated with poor prognosis. We investigated whether magnetic resonance imaging-based T1 mapping could quantify the induction and pharmacological suppression of diffuse cardiac fibrosis in murine pressure-overload hypertrophy., Methods and Results: Mice were subjected to transverse aortic constriction or sham surgery. The angiotensin receptor blocker losartan was given to half the animals. Cine-magnetic resonance imaging performed at 7 and 28 days showed hypertrophy and remodeling and systolic and diastolic dysfunction in transverse aortic constriction groups as expected. Late gadolinium-enhanced magnetic resonance imaging revealed focal signal enhancement at the inferior right ventricular insertion point of transverse aortic constriction mice concordant with the foci of fibrosis in histology. The extracellular volume fraction, calculated from pre- and postcontrast T1 measurements, was elevated by transverse aortic constriction and showed direct linear correlation with picrosirius red collagen volume fraction, thus confirming the suitability of extracellular volume fraction as an in vivo measure of diffuse fibrosis. Treatment with losartan reduced left ventricular dysfunction and prevented increased extracellular volume fraction, indicating that T1 mapping is sensitive to pharmacological prevention of fibrosis., Conclusions: Magnetic resonance imaging can detect diffuse and focal cardiac fibrosis in a clinically relevant animal model of pressure overload and is sensitive to pharmacological reduction of fibrosis by angiotensin receptor blockade. Thus, T1 mapping can be used to assess antifibrotic therapeutic strategies.

Background: Anisotropy of conduction facilitates re-entry and is, therefore, a key determinant of the stability of atrial fibrillation (AF). Little is known about the effect of AF on atrial bundle architecture and consequent changes in anisotropy of conduction and maintenance of AF., Methods and Results: Direct contact mapping was performed in left atria of goats with acute AF (n=6) or persistent AF (n=5). The degree and direction of anisotropic conduction were analyzed. Mapped tissue regions were imaged by high-resolution MRI for identification of endocardial and epicardial bundle directions. Correlation between endocardial and epicardial bundle directions and between bundle directions and anisotropic conduction was quantified. In persistent AF, epicardial bundles were oriented more perpendicularly to endocardial bundles than in acute AF (% angles<20° between epicardial and endocardial bundle directions were 7.63% and 21.25%, respectively; P<0.01). In acute AF, the direction of epicardially mapped anisotropic conduction correlated with endocardial but not with epicardial bundles. In persistent AF, the direction of anisotropic conduction correlated better with epicardial than with endocardial bundles (% angles<20° between direction of anisotropic conduction and bundle direction were 28.77% and 18.45%, respectively; P<0.01)., Conclusions: During AF, atrial bundle rearrangement manifests itself in more perpendicular orientation of epicardial to endocardial bundles. Propagation of fibrillation waves is dominated by endocardial bundles in acute AF and by epicardial bundles in persistent AF. Together with the loss of endo-epicardial electrical connections, rearrangement of atrial bundles underlies endo-epicardial dissociation of electrical activity and the development of a 3-dimensional AF substrate.

Multiple endocrine neoplasia type 1 (MEN1) is characterized by the combined occurrence of pituitary, pancreatic, and parathyroid tumors showing loss of heterozygosity in the putative tumor suppressor gene MEN1. This gene encodes the protein menin, the overexpression of which inhibits cell proliferation in vitro. In this study, we conducted a preclinical evaluation of MEN1 gene therapy in pituitary tumors of Men1(+/-) mice, using a recombinant nonreplicating adenoviral serotype 5 vector that contained the murine Men1 cDNA under control of a cytomegalovirus promoter (Men1.rAd5). Pituitary tumors in 55 Men1(+/-) female mice received a transauricular intratumoral injection of Men1.rAd5 or control treatments, followed by 5-bromo-2-deoxyuridine (BrdUrd) in drinking water for four weeks before magnetic resonance imaging (MRI) and immunohistochemical analysis. Immediate procedure-related and 4-week mortalities were similar in all groups, indicating that the adenoviral gene therapy was not associated with a higher mortality. Menin expression was higher in the Men1.rAd5-treated mice when compared with other groups. Daily proliferation rates assessed by BrdUrd incorporation were reduced significantly in Men1.rAd5-injected tumors relative to control-treated tumors. In contrast, apoptotic rates, immune T-cell response, and tumor volumes remained similar in all groups. Our findings establish that MEN1 gene replacement therapy can generate menin expression in pituitary tumors, and significantly reduce tumor cell proliferation., (©2012 AACR.)

The tumour suppressor PTEN is a key negative regulator of the PI3K-Akt pathway, and is frequently either reduced or lost in human tumours. Murine genetic studies have confirmed that reduction of Pten promotes tumourigenesis in multiple organs, and demonstrated dependency of tumour development on the activation of downstream components such as Akt. Insulin-like growth factors (IGFs) act via IGF1R to activate the PI3K-Akt pathway, and are commonly upregulated in cancer. A context-dependent interplay between IGFs and PTEN exists in normal tissue and tumours; increased IGF2 ligand supply induces Pten expression creating an autoregulatory negative feedback loop, whereas complete loss of PTEN may either cooperate with IGF overexpression in tumour promotion, or result in desensitisation to IGF ligand. However, it remains unknown whether neoplasia associated with Pten loss is dependent on upstream IGF ligand supply in vivo. We evaluated this by generation of Pten(+/-) mice with differing allelic dosage of Igf2, an imprinted gene encoding the potent embryonic and tumour growth factor Igf2. We show that biallelic Igf2 supply potentiates a previously unreported Pten(+/-) placental phenotype and results in strain-dependent cardiac hyperplasia and neonatal lethality. Importantly, we also show that the effects of Pten loss in vivo are modified by Igf2 supply, as lack of Igf2 results in extended survival and delayed tumour development while biallelic supply is associated with reduced lifespan and accelerated neoplasia in females. Furthermore, we demonstrate that reduction of PTEN protein to heterozygote levels in human MCF7 cells is associated with increased proliferation in response to IGF2, and does not result in desensitisation to IGF2 signalling. These data indicate that the effects of Pten loss at heterozygote levels commonly observed in human tumours are modified by Igf2 ligand, and emphasise the importance of the evaluation of upstream pathways in tumours with Pten loss.

Aims/hypothesis: Patients with severe gain-of-function mutations in the Kir6.2 subunit of the ATP-sensitive potassium (K(ATP)) channel, have neonatal diabetes, muscle hypotonia and mental and motor developmental delay-a condition known as iDEND syndrome. However, despite the fact that Kir6.2 forms the pore of the cardiac K(ATP) channel, patients show no obvious cardiac symptoms. The aim of this project was to use a mouse model of iDEND syndrome to determine whether iDEND mutations affect cardiac function and cardiac K(ATP) channel ATP sensitivity., Methods: We performed patch-clamp and in vivo cine-MRI studies on mice in which the most common iDEND mutation (Kir6.2-V59M) was targeted to cardiac muscle using Cre-lox technology (m-V59M mice)., Results: Patch-clamp studies of isolated cardiac myocytes revealed a markedly reduced K(ATP) channel sensitivity to MgATP inhibition in m-V59M mice (IC(50) 62 μmol/l compared with 13 μmol/l for littermate controls). In vivo cine-MRI revealed there were no gross morphological differences and no differences in heart rate, end diastolic volume, end systolic volume, stroke volume, ejection fraction, cardiac output or wall thickening between m-V59M and control hearts, either under resting conditions or under dobutamine stress., Conclusions/interpretation: The common iDEND mutation Kir6.2-V59M decreases ATP block of cardiac K(ATP) channels but was without obvious effect on heart function, suggesting that metabolic changes fail to open the mutated channel to an extent that affects function (at least in the absence of ischaemia). This may have implications for the choice of sulfonylurea used to treat neonatal diabetes.

Aims: The mdx mouse has proven to be useful in understanding the cardiomyopathy that frequently occurs in muscular dystrophy patients. Here we employed a comprehensive array of clinically relevant in vivo MRI techniques to identify early markers of cardiac dysfunction and follow disease progression in the hearts of mdx mice., Methods and Results: Serial measurements of cardiac morphology and function were made in the same group of mdx mice and controls (housed in a non-SPF facility) using MRI at 1, 3, 6, 9 and 12 months after birth. Left ventricular (LV) and right ventricular (RV) systolic and diastolic function, response to dobutamine stress and myocardial fibrosis were assessed. RV dysfunction preceded LV dysfunction, with RV end systolic volumes increased and RV ejection fractions reduced at 3 months of age. LV ejection fractions were reduced at 12 months, compared with controls. An abnormal response to dobutamine stress was identified in the RV of mdx mice as early as 1 month. Late-gadolinium-enhanced MRI identified increased levels of myocardial fibrosis in 6, 9 and 12-month-old mdx mice, the extent of fibrosis correlating with the degree of cardiac remodeling and hypertrophy., Conclusions: MRI could identify cardiac abnormalities in the RV of mdx mice as young as 1 month, and detected myocardial fibrosis at 6 months. We believe these to be the earliest MRI measurements of cardiac function reported for any mice, and the first use of late-gadolinium-enhancement in a mouse model of congenital cardiomyopathy. These techniques offer a sensitive and clinically relevant in vivo method for assessment of cardiomyopathy caused by muscular dystrophy and other diseases.

We generated a transgenic mouse model expressing the apical hypertrophic cardiomyopathy-causing mutation ACTC E99K at 50% of total heart actin and compared it with actin from patients carrying the same mutation. The actin mutation caused a higher Ca(2+) sensitivity in reconstituted thin filaments measured by in vitro motility assay (2.3-fold for mice and 1.3-fold for humans) and in skinned papillary muscle. The mutation also abolished the change in Ca(2+) sensitivity normally linked to troponin I phosphorylation. MyBP-C and troponin I phosphorylation levels were the same as controls in transgenic mice and human carrier heart samples. ACTC E99K mice exhibited a high death rate between 28 and 45 days (48% females and 22% males). At 21 weeks, the hearts of the male survivors had enlarged atria, increased interstitial fibrosis, and sarcomere disarray. MRI showed hypertrophy, predominantly at the apex of the heart. End-diastolic volume and end-diastolic pressure were increased, and relaxation rates were reduced compared with nontransgenic littermates. End-systolic pressures and volumes were unaltered. ECG abnormalities were present, and the contractile response to β-adrenergic stimulation was much reduced. Older mice (29-week-old females and 38-week-old males) developed dilated cardiomyopathy with increased end-systolic volume and continuing increased end-diastolic pressure and slower contraction and relaxation rates. ECG showed atrial flutter and frequent atrial ectopic beats at rest in some ACTC E99K mice. We propose that the ACTC E99K mutation causes higher myofibrillar Ca(2+) sensitivity that is responsible for the sudden cardiac death, apical hypertrophy, and subsequent development of heart failure in humans and mice.