Feasibility of whole-body MRI for cancer screening in children and young people with ataxia telangiectasia: A mixed methods cross-sectional study.

BACKGROUND/OBJECTIVES: Ataxia telangiectasia (A-T) is an inherited multisystem disorder with increased sensitivity to ionising radiation and elevated cancer risk. Although other cancer predisposition syndromes have established cancer screening protocols, evidence-based guidelines for cancer screening in A-T are lacking. This study sought to assess feasibility of a cancer screening protocol based on whole-body MRI (WB-MRI) in children and young people with A-T. DESIGN/METHODS: Children and young people with A-T were invited to undergo a one-off non-sedated 3-Tesla WB-MRI. Completion rate of WB-MRI was recorded and diagnostic image quality assessed by two experienced radiologists, with pre-specified success thresholds for scan completion of >50% participants and image quality between acceptable to excellent in 65% participants. Positive imaging findings were classified according to the ONCO-RADS system. Post-participation interviews were performed with recruited families to assess the experience of participating and feelings about waiting for, and communication of, the findings of the scan. RESULTS: Forty-six children and young people with A-T were identified, of which 36 were eligible to participate, 18 were recruited and 16 underwent WB-MRI. Nineteen parents participated in interviews. Fifteen participants (83%) completed the full WB-MRI scan protocol. The pre-specified image quality criterion was achieved with diagnostic images obtained in at least 93% of each MRI sequence. Non-malignant scan findings were present in 4 (25%) participants. Six themes were identified from the interviews: (1) anxiety is a familiar feeling, (2) the process of MRI scanning is challenging for some children and families, (3) preparation is essential to reduce stress, (4) WB-MRI provides the reassurance about the physical health that families need, (5) WB-MRI experience turned out to be a positive experience and (6) WB-MRI allows families to be proactive. CONCLUSION: This study shows that WB-MRI for cancer screening is feasible and well-accepted by children and young people with A-T and their families.

Transient pacing in pigs with complete heart block via myocardial injection of mRNA coding for the T-box transcription factor 18.

The adenovirus-mediated somatic transfer of the embryonic T-box transcription factor 18 (TBX18) gene can convert chamber cardiomyocytes into induced pacemaker cells. However, the translation of therapeutic TBX18-induced cardiac pacing faces safety challenges. Here we show that the myocardial expression of synthetic TBX18 mRNA in animals generates de novo pacing and limits innate and inflammatory immune responses. In rats, intramyocardially injected mRNA remained localized, whereas direct myocardial injection of an adenovirus carrying a reporter gene resulted in diffuse expression and in substantial spillover to the liver, spleen and lungs. Transient expression of TBX18 mRNA in rats led to de novo automaticity and pacemaker properties and, compared with the injection of adenovirus, to substantial reductions in the expression of inflammatory genes and in activated macrophage populations. In rodent and clinically relevant porcine models of complete heart block, intramyocardially injected TBX18 mRNA provided rate-adaptive cardiac pacing for one month that strongly correlated with the animal's sinus rhythm and physical activity. TBX18 mRNA may aid the development of biological pacemakers.

Whole-body MRI for cancer surveillance in ataxia-telangiectasia: A qualitative study of the perspectives of people affected by A-T and their families.

BACKGROUND/OBJECTIVES: Ataxia-telangiectasia (A-T) is a complex inherited disease associated with an increased risk of malignancy. Surveillance guidelines have demonstrated significant health benefits in other cancer predisposition syndromes. However, evidence-based guidelines for cancer screening are not currently used in the United Kingdom for people affected by A-T. This study aims to understand how people with A-T and their parents feel about cancer surveillance using whole-body magnetic resonance imaging (MRI) to inform the future development of cancer surveillance guidelines. DESIGN/METHODS: We conducted semistructured interviews with people affected by A-T. Data were analysed inductively using thematic analysis. RESULTS: Nine parents of children with A-T and four adults with A-T were interviewed. Five main themes emerged from the data, including (1) cancer screening was considered invaluable with the perceived value of early detection highlighted; (2) the cancer fear can increase anxiety; (3) the perceived limitations around current practice, with the responsibility for monitoring falling too strongly on parents and patients; (4) the need for effective preparation for cancer screening, including clear communication and (5) the challenges associated with MRI screening, where specific recommendations were made for improving the child's experience. CONCLUSION: This study suggests that stakeholders are positive about the perceived advantages of a cancer screening programme. Ongoing support and preparation techniques should be adopted to maximise adherence and minimise adverse psychosocial outcomes. PATIENT OR PUBLIC CONTRIBUTION: People with A-T and parents of people with A-T were actively involved in this study by giving their consent to be interviewed. An independent parent representative contributed to the study, supporting the research team in interpreting and commenting on the appropriateness of the language used in this report.

DNA Tension Probes Show that Cardiomyocyte Maturation Is Sensitive to the Piconewton Traction Forces Transmitted by Integrins.

Cardiac muscle cells (CMCs) are the unit cells that comprise the heart. CMCs go through different stages of differentiation and maturation pathways to fully mature into beating cells. These cells can sense and respond to mechanical cues through receptors such as integrins which influence maturation pathways. For example, cell traction forces are important for the differentiation and development of functional CMCs, as CMCs cultured on varying substrate stiffness function differently. Most work in this area has focused on understanding the role of bulk extracellular matrix stiffness in mediating the functional fate of CMCs. Given that stiffness sensing mechanisms are mediated by individual integrin receptors, an important question in this area pertains to the specific magnitude of integrin piconewton (pN) forces that can trigger CMC functional maturation. To address this knowledge gap, we used DNA adhesion tethers that rupture at specific thresholds of force (∼12, ∼56, and ∼160 pN) to test whether capping peak integrin tension to specific magnitudes affects CMC function. We show that adhesion tethers with greater force tolerance lead to functionally mature CMCs as determined by morphology, twitching frequency, transient calcium flux measurements, and protein expression (F-actin, vinculin, α-actinin, YAP, and SERCA2a). Additionally, sarcomeric actinin alignment and multinucleation were significantly enhanced as the mechanical tolerance of integrin tethers was increased. Taken together, the results show that CMCs harness defined pN integrin forces to influence early stage development. This study represents an important step toward biophysical characterization of the contribution of pN forces in early stage cardiac differentiation.

Engineered Cardiac Pacemaker Nodes Created by TBX18 Gene Transfer Overcome Source-Sink Mismatch.

Every heartbeat originates from a tiny tissue in the heart called the sinoatrial node (SAN). The SAN harbors only ≈10 000 cardiac pacemaker cells, initiating an electrical impulse that captures the entire heart, consisting of billions of cardiomyocytes for each cardiac contraction. How these rare cardiac pacemaker cells (the electrical source) can overcome the electrically hyperpolarizing and quiescent myocardium (the electrical sink) is incompletely understood. Due to the scarcity of native pacemaker cells, this concept of source-sink mismatch cannot be tested directly with live cardiac tissue constructs. By exploiting TBX18 induced pacemaker cells by somatic gene transfer, 3D cardiac pacemaker spheroids can be tissue-engineered. The TBX18 induced pacemakers (sphTBX18) pace autonomously and drive the contraction of neighboring myocardium in vitro. TBX18 spheroids demonstrate the need for reduced electrical coupling and physical separation from the neighboring ventricular myocytes, successfully recapitulating a key design principle of the native SAN. ß-Adrenergic stimulation as well as electrical uncoupling significantly increase sphTBX18s' ability to pace-and-drive the neighboring myocardium. This model represents the first platform to test design principles of the SAN for mechanistic understanding and to better engineer biological pacemakers for therapeutic translation.

Induced cardiac pacemaker cells survive metabolic stress owing to their low metabolic demand.

Cardiac pacemaker cells of the sinoatrial node initiate each and every heartbeat. Compared with our understanding of the constituents of their electrical excitation, little is known about the metabolic underpinnings that drive the automaticity of pacemaker myocytes. This lack is largely owing to the scarcity of native cardiac pacemaker myocytes. Here, we take advantage of induced pacemaker myocytes generated by TBX18-mediated reprogramming (TBX18-iPMs) to investigate comparative differences in the metabolic program between pacemaker myocytes and working cardiomyocytes. TBX18-iPMs were more resistant to metabolic stresses, exhibiting higher cell viability upon oxidative stress. TBX18-induced pacemaker myocytes (iPMs) expensed a lower degree of oxidative phosphorylation and displayed a smaller capacity for glycolysis compared with control ventricular myocytes. Furthermore, the mitochondria were smaller in TBX18-iPMs than in the control. We reasoned that a shift in the balance between mitochondrial fusion and fission was responsible for the smaller mitochondria observed in TBX18-iPMs. We identified a mitochondrial inner membrane fusion protein, Opa1, as one of the key mediators of this process and demonstrated that the suppression of Opa1 expression increases the rate of synchronous automaticity in TBX18-iPMs. Taken together, our data demonstrate that TBX18-iPMs exhibit a low metabolic demand that matches their mitochondrial morphology and ability to withstand metabolic insult.

A rat model of complete atrioventricular block recapitulates clinical indices of bradycardia and provides a platform to test disease-modifying therapies.

Complete atrioventricular block (CAVB) is a life-threatening arrhythmia. A small animal model of chronic CAVB that properly reflects clinical indices of bradycardia would accelerate the understanding of disease progression and pathophysiology, and the development of therapeutic strategies. We sought to develop a surgical model of CAVB in adult rats, which could recapitulate structural remodeling and arrhythmogenicity expected in chronic CAVB. Upon right thoracotomy, we delivered electrosurgical energy subepicardially via a thin needle into the atrioventricular node (AVN) region of adult rats to create complete AV block. The chronic CAVB animals developed dilated and hypertrophied ventricles with preserved systolic functions due to compensatory hemodynamic remodeling. Ventricular tachyarrhythmias, which are difficult to induce in the healthy rodent heart, could be induced upon programmed electrical stimulation in chronic CAVB rats and worsened when combined with ß-adrenergic stimulation. Focal somatic gene transfer of TBX18 to the left ventricular apex in the CAVB rats resulted in ectopic ventricular beats within days, achieving a de novo ventricular rate faster than the slow atrioventricular (AV) junctional escape rhythm observed in control CAVB animals. The model offers new opportunities to test therapeutic approaches to treat chronic and severe CAVB which have previously only been testable in large animal models.

Strength-duration relationship as a tool to prioritize cardiac tissue properties that govern electrical excitability.

Engineered cardiac tissue and cardiomyocyte cell cultures offer wide opportunities for improved therapeutic intervention and laboratory heart models. Electrical field excitation is a common intervention in the production of engineered tissue and the investigation of the electrical properties of in vitro cell cultures. In this work, we use strength-duration relationships to investigate systematically factors influencing electrical excitability of two- (2D) and three-dimensional (3D) neonatal rat ventricular myocyte cultures. We find that the strength of the voltage pulse is negatively correlated with the threshold duration, as predicted by the Lapicque-Hill equation, and show that higher pacing frequencies require higher thresholds to capture paced cultures. We also study the impact of properties intrinsic to the 2D and 3D cultures on strength-duration relationships. We show that a smaller culture dimension, perpendicular anisotropic culture orientation with respect to electrical field, higher proportion of added fibroblasts, and TBX18-induced pacemaker reprogramming independently result in higher stimulation thresholds. These properties reflect the characteristics of the well-insulated endogenous pacemaking tissue in the heart (sinoatrial node) and should guide the engineering of biological pacemakers for improved outcomes. NEW & NOTEWORTHY Gaps exist in the availability of in vitro functional assessment tools that can emulate the integration of regenerative cells and tissues to the host myocardium. We use strength-duration relationships of electrically stimulated two- and three-dimensional myocardial constructs to study the effects of pacing frequency, culture dimensions, anisotropic cell alignment, fibroblast content, and pacemaker phenotype on electrical excitability. Our study delivers electrical strength-duration as a quantifiable parameter to evaluate design parameters of engineered cardiac tissue constructs.

Assessment of micro-mechanical variations in experimental arteriovenous fistulae using atomic force microscopy.

PURPOSE: This study presents a method to quantify micro-stiffness variations in experimental arteriovenous fistulae (AVF). METHODS: AVF created by anastomosing the superficial epigastric vein to the femoral artery in Sprague-Dawley rats were allowed to remodel for 21 days before being harvested and preserved in culture medium. A custom atomic force microscope was used to measure microvascular stiffness (Young's modulus) in three areas of the AVF: the inflow artery, the juxta-anastomotic area, and the outflow vein. Morphometric measurements and collagen and elastin contents were also determined. RESULTS: Atomic force microscopy indentation revealed an increased stiffness in the juxta-anastomotic area of the AVF compared to the outflow vein and inflow artery. The juxta-anastomotic area was also significantly stiffer than the contralateral vein. The lack of elasticity (higher Young's modulus) of the juxta-anastomotic region was associated with a thicker vascular wall that was rich in collagen but poor in elastin. CONCLUSIONS: This study demonstrates for the first time the feasibility of using atomic force microscopy to measure local stiffness variations in experimental AVF. This technique could be instrumental in advancing our understanding of how micro-spatial organization of the AVF wall determines the overall biomechanical performance of this type of vascular access.

Pre-existing and Postoperative Intimal Hyperplasia and Arteriovenous Fistula Outcomes.

BACKGROUND: The contribution of intimal hyperplasia (IH) to arteriovenous fistula (AVF) failure is uncertain. This observational study assessed the relationship between pre-existing, postoperative, and change in IH over time and AVF outcomes. STUDY DESIGN: Prospective cohort study with longitudinal assessment of IH at the time of AVF creation (pre-existing) and transposition (postoperative). Patients were followed up for up to 3.3 years. SETTING & PARTICIPANTS: 96 patients from a single center who underwent AVF surgery initially planned as a 2-stage procedure. Veins and AVF samples were collected from 66 and 86 patients, respectively. Matched-pair tissues were available from 56 of these patients. PREDICTORS: Pre-existing, postoperative, and change in IH over time. OUTCOMES: Anatomic maturation failure was defined as an AVF that never reached a diameter > 6mm. Primary unassisted patency was defined as the time elapsed from the second-stage surgery to the first intervention. MEASUREMENTS: Maximal intimal thickness in veins and AVFs and change in intimal thickness over time. RESULTS: Pre-existing IH (>0.05mm) was present in 98% of patients. In this group, the median intimal thickness increased 4.40-fold (IQR, 2.17- to 4.94-fold) between AVF creation and transposition. However, this change was not associated with pre-existing thickness (r(2)=0.002; P=0.7). Ten of 96 (10%) AVFs never achieved maturation, whereas 70% of vascular accesses remained patent at the end of the observational period. Postoperative IH was not associated with anatomic maturation failure using univariate logistic regression. Pre-existing, postoperative, and change in IH over time had no effects on primary unassisted patency. LIMITATIONS: The small number of patients from whom longitudinal tissue samples were available and low incidence of anatomic maturation failure, which decreased the statistical power to find associations between end points and IH. CONCLUSIONS: Pre-existing, postoperative, and change in IH over time were not associated with 2-stage AVF outcomes.

Periodontal ligament cells cultured under steady-flow environments demonstrate potential for use in heart valve tissue engineering.

A major drawback of mechanical and prosthetic heart valves is their inability to permit somatic growth. By contrast, tissue-engineered pulmonary valves potentially have the capacity to remodel and integrate with the patient. For this purpose, adult stem cells may be suitable. Previously, human periodontal ligament cells (PDLs) have been explored as a reliable and robust progenitor cell source for cardiac muscle regeneration (Pelaez, D. Electronic Thesis and Dissertation Database, Coral Gables, FL, May 2011). Here, we investigate the potential of PDLs to support the valve lineage, specifically the concomitant differentiation to both endothelial cell (EC) and smooth muscle cell (SMC) types. We were able to successfully promote PDL differentiation to both SMC and EC phenotypes through a combination of stimulatory approaches using biochemical and mechanical flow conditioning (steady shear stress of 1 dyne/cm(2)), with flow-based mechanical conditioning having a predominant effect on PDL differentiation, particularly to ECs; in addition, strong expression of the marker FZD2 and an absence of the marker MLC1F point toward a unique manifestation of smooth muscle by PDLs after undergoing steady-flow mechanical conditioning alone, possible by only the heart valve and pericardium phenotypes. It was also determined that steady flow (which was performed using a physiologically relevant [for heart valves] magnitude of ~5-6 dynes/cm(2)) augmented the synthesis of the extracellular matrix collagen proteins. We conclude that under steady-flow dynamic culture environments, human PDLs can differentiate to heterogeneous cell populations that are relevant to heart valve tissue engineering. Further exploration of human PDLs for this purpose is thus warranted.