Cardiac Imaging for Regenerative Therapy and Tissue Engineering

Cardiovascular disease remains the number 1 cause of death worldwide. Over the past 20 years, therapies for treating cardiac disease have come of age and coronary heart disease in particular has seen a revolution in new treatments such as statins, stents and beta blockers. These therapies have slowed death rates and have shown potential to minimise ischemia induced atrophy following myocardial infarction. Crucially however, they are unable to recover lost heart function due to cardiomyocyte death, resulting in poor prognosis for patients. Myocardial regeneration therapy is a new strategy towards treating cardiac disease that engrafts regenerative cells and biomaterials to the myocardium to stimulate repair of tissue and restore contractile function. Cardiac regeneration therapy has made a rapid translation from preclinical research to clinical trials with the first trial in humans published in 2001. Clinical trials in the years since however have produced underwhelming results and there is a general consensus that further preclinical optimisation with powerful non-invasive imaging data will be key to the future success of regenerative medicine in humans. Magnetic resonance imaging is unparalleled in providing non-invasive multiparametric imaging of both global and regional cardiac structure and function. MRI provides high spatiotemporal resolution and multiple contrast mechanisms revealing information about molecular changes in the myocardium. These imaging abilities make MRI a versatile and powerful tool in the preclinical optimisation of cardiac regeneration therapies. Over the chapters presented in this thesis I have established a set of MR imaging techniques that enable valuable in vivo characterisation of cardiac function and structure in for use in studies of regenerative therapy. It is hoped that the methods developed over the course of this thesis aid in the uptake of imaging applications in studies of regenerative medicine and that the wide range of imaging tools demonstrated help to bring regenerative medicine closer to practical clinical therapy

A new look at sodium channel β subunits.

Voltage-gated sodium (Nav) channels are intrinsic plasma membrane proteins that initiate the action potential in electrically excitable cells. They are a major focus of research in neurobiology, structural biology, membrane biology and pharmacology. Mutations in Nav channels are implicated in a wide variety of inherited pathologies, including cardiac conduction diseases, myotonic conditions, epilepsy and chronic pain syndromes. Drugs active against Nav channels are used as local anaesthetics, anti-arrhythmics, analgesics and anti-convulsants. The Nav channels are composed of a pore-forming α subunit and associated β subunits. The β subunits are members of the immunoglobulin (Ig) domain family of cell-adhesion molecules. They modulate multiple aspects of Nav channel behaviour and play critical roles in controlling neuronal excitability. The recently published atomic resolution structures of the human β3 and β4 subunit Ig domains open a new chapter in the study of these molecules. In particular, the discovery that β3 subunits form trimers suggests that Nav channel oligomerization may contribute to the functional properties of some β subunits

Multi-modal hydrogel-based platform to deliver and monitor cardiac progenitor/stem cell engraftment

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

Effectiveness of training stop-smoking advisers to deliver cessation support to the UK national proposed standard versus usual care in Malaysia: a two-arm cluster randomised controlled trial

AIMS: To assess the effectiveness of training stop smoking services providers in Malaysia to deliver support for smoking cessation based on the UK National Centre for Smoking Cessation and Training (NCSCT) standard treatment programme compared with usual care. DESIGN: Two‐arm cluster‐randomized controlled effectiveness trial across 19 sites with follow‐up at 4‐week, 3‐month, and 6‐month. SETTING: Stop smoking services operating in public hospitals in Malaysia. PARTICIPANTS: Five hundred and two smokers [mean ± standard deviation (SD), age 45.6 (13.4) years; 97.4% male] attending stop smoking services in hospital settings in Malaysia: 330 in 10 hospitals in the intervention condition and 172 in nine hospitals in the control condition. INTERVENTION AND COMPARATOR: The intervention consisted of training stop‐smoking practitioners to deliver support and follow‐up according to the NCSCT Standard Treatment Programme. The comparator was usual care (brief support and follow‐up). MEASUREMENTS: The primary outcome was continuous tobacco smoking abstinence up to 6 months in smokers who received smoking cessation treatment, verified by expired‐air carbon monoxide (CO) concentration. Secondary outcomes were continuous CO‐verified tobacco smoking abstinence up to 4 weeks and 3 months. RESULTS: Follow‐up rates at 4 weeks, 3 months and 6 months were 80.0, 70.6 and 53.3%, respectively, in the intervention group and 48.8, 30.8 and 23.3%, respectively, in the control group. At 6‐month follow‐up, 93 participants in the intervention group and 19 participants in the control group were abstinent from smoking, representing 28.2 versus 11.0% in an intention‐to‐treat (ITT) analysis assuming that participants with missing data had resumed smoking, and 52.8 versus 47.5% in a follow‐up‐only (FUO) analysis. Unadjusted odds ratios (accounting for clustering) were 5.04, (95% confidence interval (CI) = 1.22–20.77, P = 0.025) and 1.70, (95% CI = 0.25–11.53, P = 0.589) in the ITT and FUO analyses, respectively. Abstinence rates at 4 week and 3 month follow‐ups were significantly higher in the intervention versus control group in the ITT but not the FUO analysis. CONCLUSIONS: On an intention‐to‐treat analysis with missing‐equals‐smoking imputation, training Malaysian stop smoking service providers in the UK National Centre for Smoking Cessation and Training standard treatment programme appeared to increase 6 month continuous abstinence rates in smokers seeking help with stopping compared with usual care. However, the effect may have been due to increasing follow‐up rates

Non-invasive MRI biomarkers for the early assessment of iron overload in a humanized mouse model of β-thalassemia

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

Myocardial Viability Imaging using Manganese-Enhanced MRI in the First Hours after Myocardial Infarction

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 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 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 -based contrast agents open the possibility of using MEMRI for direct evaluation of myocardial viability early after ischemic onset in patients

Gating control of the cardiac sodium channel Nav1.5 by its β3-subunit involves distinct roles for a transmembrane glutamic acid and the extracellular domain.

The auxiliary β3-subunit is an important functional regulator of the cardiac sodium channel Nav1.5, and some β3 mutations predispose individuals to cardiac arrhythmias. The β3-subunit uses its transmembrane α-helix and extracellular domain to bind to Nav1.5. Here, we investigated the role of an unusually located and highly conserved glutamic acid (Glu-176) within the β3 transmembrane region and its potential for functionally synergizing with the β3 extracellular domain (ECD). We substituted Glu-176 with lysine (E176K) in the WT β3-subunit and in a β3-subunit lacking the ECD. Patch-clamp experiments indicated that the E176K substitution does not affect the previously observed β3-dependent depolarizing shift of V½ of steady-state inactivation but does attenuate the accelerated recovery from inactivation conferred by the WT β3-subunit. Removal of the β3-ECD abrogated both the depolarizing shift of steady-state inactivation and the accelerated recovery, irrespective of the presence or absence of the Glu-176 residue. We found that steady-state inactivation and recovery from inactivation involve movements of the S4 helices within the DIII and DIV voltage sensors in response to membrane potential changes. Voltage-clamp fluorometry revealed that the E176K substitution alters DIII voltage sensor dynamics without affecting DIV. In contrast, removal of the ECD significantly altered the dynamics of both DIII and DIV. These results imply distinct roles for the β3-Glu-176 residue and the β3-ECD in regulating the conformational changes of the voltage sensors that determine channel inactivation and recovery from inactivation

Assessment of protein allergenicity on the basis of immune reactivity: animal models.

Because of the public concern surrounding the issue of the safety of genetically modified organisms, it is critical to have appropriate methodologies to aid investigators in identifying potential hazards associated with consumption of foods produced with these materials. A recent panel of experts convened by the Food and Agriculture Organization and World Health Organization suggested there is scientific evidence that using data from animal studies will contribute important information regarding the allergenicity of foods derived from biotechnology. This view has given further impetus to the development of suitable animal models for allergenicity assessment. This article is a review of what has been achieved and what still has to be accomplished regarding several different animal models. Progress made in the design and evaluation of models in the rat, the mouse, the dog and in swine is reviewed and discussed