Biology subjects

Trafford, A.

Publications and source records attributed to Trafford, A..

6 recordsLinked to original sources

bioRxiv2026

Cardiac microtubules mediate transverse (t)-tubule growth and homeostasis

Transverse (t)-tubules enable rapid, synchronous Ca release required for efficient cardiac contraction by bringing L-type Ca channels into close apposition with ryanodine receptors. In heart failure with reduced ejection fraction (HFrEF), t-tubule disorganisation and loss occur alongside cardiac microtubule remodelling, contributing to impaired Ca handling and contractile dysfunction. Despite their canonical function in contraction, how t-tubules develop is unknown. Microtubules support delivery of L-type Ca channels to t-tubules via Amphiphysin-II/BIN1, yet whether microtubules directly regulate t-tubule formation and maintenance is unclear. Here, we investigated a role for microtubules in t-tubule development and homeostasis. Neonatal rat ventricular myocytes (NRVMs), which lack endogenous t-tubules, were used as a reductionist model in which BIN1 overexpression induces nascent membrane tubules. Microtubule depolymerisation with nocodazole before BIN1 overexpression impaired BIN1-driven tubule formation, reducing tubule density and length. Dynein inhibition with EHNA produced similar effects, indicating a requirement for microtubule-based motor activity during tubule elongation. Knockdown of the microtubule +TIP tracking protein CLIP-170 also reduced BIN1-driven tubule density, implicating BIN1-CLIP-170-dependent microtubule capture in tubule initiation. Microtubules were also required to maintain existing tubules. In NRVMs with established BIN1-driven tubules, microtubule depolymerisation, microtubule stabilisation or dynein inhibition each reduced tubule density and length. Consistent with this, acute microtubule depolymerisation or stabilisation disrupted native t-tubule networks in isolated adult sheep left atrial myocytes. Together, these findings identify cardiac microtubules as active regulators of t-tubule architecture. We propose that BIN1-dependent tubule formation requires CLIP-170-mediated microtubule plus-end capture and dynein-dependent elongation, while ongoing microtubule dynamics are necessary to preserve mature t-tubule structure.

physiology↗

bioRxiv2026

Developmental Hypoxia Increases Susceptibility to Cardiac Ventricular Arrhythmias in Adult Offspring

Ventricular arrhythmias are the leading cause of sudden cardiac death. It is well-established that environmental factors contribute to the origin and penetrance of ventricular arrhythmic disorders. However, to our knowledge, no studies have considered the role of the intrauterine environment. In this study, we investigated the long-term effects of fetal hypoxia on ventricular arrhythmia susceptibility. Pregnant Wistar rats were assigned to normoxia (21% O2) or hypoxia (13% O2 between gestational days 6-20), and offspring were raised to 6 months. Hearts were isolated and loaded with the fluorescent calcium- and voltage-sensitive indicators, Rhod-2 and RH237, respectively. Optical mapping was performed while the left ventricle was burst paced (10-20hz) to induce arrhythmias. Hearts isolated from adult offspring exposed to fetal hypoxia were more susceptible to arrythmia during burst pacing, compared to controls. This phenotype was associated with prolonged Ca2+ transients and action potentials, an increased frequency of Ca2+ waves and delayed after depolarisations, as well as lower gene and protein expression of the sarcoplasmic reticulum Ca2+ ATPase. Collectively, our data shows that fetal hypoxia can programme ventricular arrhythmia sensitivity in adulthood, driven by abnormalities in excitation-contraction coupling. This is the first evidence that some ventricular arrhythmias may have a developmental origin, highlighting pregnancy as a potential window for early preventive intervention.

developmental biology↗

bioRxiv2024

Acute sildenafil administration reduces susceptibility to induced atrial fibrillation in sheep

BackgroundSildenafil is a PDE5 inhibitor with a very good safety profile and animal models suggest it may be beneficial in the treatment of heart failure and ventricular fibrillation. Sildenafil has also been associated with a reduced incidence of atrial fibrillation (AF) in a retrospective observational study. We have therefore sought to determine whether sildenafil has a direct effect on atrial electrophysiology and resultant AF burden. MethodsInvasive electrophysiological studies were performed in 12 anaesthetised healthy adult female Welsh mountain sheep. Pacing protocols were performed in the right atrium before and after administration of an acute 10 mg intravenous bolus of sildenafil and the burden of AF assessed. ResultsSildenafil profoundly reduced the vulnerability to AF, decreasing AF duration (112.2 {+/-} 73.5 s vs. 3.3 {+/-} 1.4 s), the number of burst pacing inductions causing AF (90 % vs 70 %) and the complexity of AF. The antiarrhythmic effects of sildenafil were determined to be resultant of prolongation of both the atrial effective refractory period (146.9 {+/-} 7.2 ms vs 166.2 {+/-} 32.5 ms) and the atrial excitation wavelength (12.9 {+/-} 0.07 cm vs 15.0 {+/-} 0.07 cm) and resulted in a shallower restitution curve, reflected in a decreased magnitude of monophasic action potential alternans (0.09 {+/-} 0.001 mV vs 0.05 {+/-} 0.10 mV). ConclusionsIn the subjectively healthy atria of a highly translational model a strong antiarrhythmic effect upon acute sildenafil application was observed suggestive of a potential clinical benefit in AF.

physiology↗

bioRxiv2023

A novel minimally invasive and reproducible large animal ischaemia-reperfusion-infarction model: methodology and model validation

Ischaemic heart disease remains a leading cause of premature mortality and morbidity. Understanding the associated pathophysiological mechanisms of cardiac dysfunction arising from ischaemic heart disease and the identification of sites of novel therapeutic intervention requires a preclinical model that reproduces the key clinical characteristics of myocardial ischaemia, reperfusion and infarction. Here we describe and validate a refined and minimally invasive translationally relevant approach to induce ischaemia, reperfusion and infarction in the sheep. The protocol uses clinical cardiology devices and approaches and would be readily adopted by researchers with access to standard fluoroscopic instrumentation. In addition to being minimally invasive, the major refinements associated with the described methodology are the implantation of an intracardiac defibrillator prior to coronary engagement and use of an antiarrhythmic medication protocol during the procedure. These refinements lead to a reduction of intraoperative mortality to 6.7 %. The model produces key characteristics associated with the 4th Universal Definition of Myocardial Infarction including electrocardiographic changes, elevated cardiac biomarkers and cardiac wall motility defects. In conclusion, the model closely replicates the clinical paradigm of myocardial ischaemia, reperfusion and infarction in a translationally relevant large-animal setting and the applied refinements reduce the incidence of intraoperative mortality typically associated with preclinical myocardial infarction models.

physiology↗

bioRxiv2022

Peptide location fingerprinting identifies species- and tissue-conserved structural remodelling of proteins as a consequence of ageing and disease

Extracellular matrix (ECM) in the intervertebral disc (IVD), lung and artery are thought to undergo the age-dependant accumulation of damage by chronic exposure to mechanisms such as reactive oxygen species, proteases and glycation. It is unknown whether this damage accumulation is species-dependant (via differing lifespans and hence cumulative exposures) or whether it can influence the progression of age-related diseases such as atherosclerosis. Peptide location fingerprinting (PLF) is a new proteomic analysis method, capable of the non-targeted identification of structure-associated changes within proteins. Here we applied PLF to publicly available ageing human IVD (outer annulus fibrosus), ageing mouse lung and human arterial atherosclerosis datasets and identified novel target proteins alongside common age-associated differences within protein structures which were conserved between tissue regions, organs, sexes and species and in age-related disease. We identify peptide yield differences across protein structures which coincide with biological regions, potentially reflecting the functional consequences of ageing or atherosclerosis for macromolecular assemblies (collagen VI and fibrin), enzyme/inhibitor activity (cathepsin B and alpha-2 macroglobulin), activation states (complement C3 and thrombin) and interaction states (laminins, perlecan, fibronectin, filamin-A, collagen XIV and apolipoprotein-B). Furthermore, we show that alpha-2 macroglobulin, prothrombin, collagen XIV and apolipoprotein-B all exhibit possible shared structural consequences in IVD ageing and arterial atherosclerosis, providing novel links between an age-related disease and intrinsic ageing. Crucially, we also demonstrate that fibronectin, laminin beta chains and filamin-A all exhibit conserved age-associated structural differences between mouse lung and human IVD, providing evidence that ECM, and their associating proteins, may be subjected to potentially similar mechanisms or consequences of ageing across species, irrespective of differences in lifespan and tissue function.

molecular biology↗

bioRxiv2020

Mechanistic insights into ventricular arrhythmogenesis of hydroxychloroquine and azithromycin for the treatment of COVID-19

Background: Recent reports on the use of hydroxychloroquine (HCQ) alone, or combined with azithromycin (AZM) in the management of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have raised cardiac safety concerns. Currently, there is limited mechanistic data evaluating cardiac safety with HCQ and AZM therapy. Methods: Using comprehensive In Vitro ProArrhythmia Assay (CiPA) Schema IC50 paradigms, we examined the cardiac electrophysiological effects of HCQ and HCQ/AZM. Molecular modelling explored HCQ and AZM binding properties to hERG. Langendorff-perfused guinea-pig hearts were electrically and optically mapped by multi-electrode array and voltage (RH237) and Ca2+ (Rhod-2 AM) dyes. Human action potential and ion current reconstructions were performed in silico. Results: HCQ blocked IKr and IK1 with IC50 concentrations (10+-0.6 and 34+-5.0 microM) within the therapeutic range observed clinically. HCQ also blocked INa and ICaL but at higher IC50, whilst Ito and IKs were unaffected. Contrastingly, AZM produced minor inhibition of INa, ICaL, IKs, and IKr,, with no effect on IK1 and Ito. HCQ + AZM combined inhibited IKr and IK1 with IC50s of 7.7 +- 0.8 microM and 30.4 +- 3.0 microM, but spared INa, ICaL and Ito,. Molecular modelling confirmed potential HCQ binding to hERG. Cardiac mapping and ECG studies in isolated hearts demonstrated that HCQ slowed heart rate and ventricular conduction with associated prolongation of PR, QRS and QT intervals. Optical mapping demonstrated, and prolonged, more heterogeneous, action potential durations and intracellular Ca2+ transients. These effects were accentuated with combined HCQ+AZM treatment, which elicited electrical alternans, re-entrant circuits and wave breaks. Reconstruction in a human in-silico model demonstrated that this is attributable to the integrated action of HCQ and AZM reducing IKr, IKs and IK1. Conclusions: These data provide an electrophysiological basis for recent FDA guidelines cautioning against combined HCQ/AZM administration for the treatment of Covid-19 on the grounds of potential cardiac safety. We would strongly recommend monitoring of electrocardiographic QT interval with the use of this combination of medications.Competing Interest StatementThe authors have declared no competing interest.

pharmacology and toxicology↗