Mesoscale standing wave imaging.

Standing wave (SW) microscopy is a method that uses an interference pattern to excite fluorescence from labelled cellular structures and produces high-resolution images of three-dimensional objects in a two-dimensional dataset. SW microscopy is performed with high-magnification, high-numerical aperture objective lenses, and while this results in high-resolution images, the field of view is very small. Here we report upscaling of this interference imaging method from the microscale to the mesoscale using the Mesolens, which has the unusual combination of a low-magnification and high-numerical aperture. With this method, we produce SW images within a field of view of 4.4 mm × 3.0 mm that can readily accommodate over 16,000 cells in a single dataset. We demonstrate the method using both single-wavelength excitation and the multi-wavelength SW method TartanSW. We show application of the method for imaging of fixed and living cells specimens, with the first application of SW imaging to study cells under flow conditions.

Impact of vaccination on hospitalization and mortality from COVID-19 in patients with primary and secondary immunodeficiency: The United Kingdom experience.

Background: Individuals with primary and secondary immunodeficiency (PID/SID) were shown to be at risk of poor outcomes during the early stages of the SARS-CoV-2 pandemic. SARS-CoV-2 vaccines demonstrate reduced immunogenicity in these patients. Objectives: To understand whether the risk of severe COVID-19 in individuals with PID or SID has changed following the deployment of vaccination and therapeutics in the context of the emergence of novel viral variants of concern. Methods: The outcomes of two cohorts of patients with PID and SID were compared: the first, infected between March and July 2020, prior to vaccination and treatments, the second after these intervention became available between January 2021 and April 2022. Results: 22.7% of immunodeficient patients have been infected at least once with SARS-CoV-2 since the start of the pandemic, compared to over 70% of the general population. Immunodeficient patients were typically infected later in the pandemic when the B.1.1.529 (Omicron) variant was dominant. This delay was associated with receipt of more vaccine doses and higher pre-infection seroprevalence. Compared to March-July 2020, hospitalization rates (53.3% vs 17.9%, p<0.0001) and mortality (Infection fatality rate 20.0% vs 3.4%, p=0.0003) have significantly reduced for patients with PID but remain elevated compared to the general population. The presence of a serological response to vaccination was associated with a reduced duration of viral detection by PCR in the nasopharynx. Early outpatient treatment with antivirals or monoclonal antibodies reduced hospitalization during the Omicron wave. Conclusions: Most individuals with immunodeficiency in the United Kingdom remain SARS-CoV-2 infection naïve. Vaccination, widespread availability of outpatient treatments and, possibly, the emergence of the B.1.1.529 variant have led to significant improvements in morbidity and mortality followings SARS-CoV-2 infection since the start of the pandemic. However, individuals with PID and SID remain at significantly increased risk of poor outcomes compared to the general population; mitigation, vaccination and treatment strategies must be optimized to minimize the ongoing burden of the pandemic in these vulnerable cohorts.

Outcomes following SARS-CoV-2 infection in patients with primary and secondary immunodeficiency in the UK.

In March 2020, the United Kingdom Primary Immunodeficiency Network (UKPIN) established a registry of cases to collate the outcomes of individuals with PID and SID following SARS-CoV-2 infection and treatment. A total of 310 cases of SARS-CoV-2 infection in individuals with PID or SID have now been reported in the UK. The overall mortality within the cohort was 17.7% (n = 55/310). Individuals with CVID demonstrated an infection fatality rate (IFR) of 18.3% (n = 17/93), individuals with PID receiving IgRT had an IFR of 16.3% (n = 26/159) and individuals with SID, an IFR of 27.2% (n = 25/92). Individuals with PID and SID had higher inpatient mortality and died at a younger age than the general population. Increasing age, low pre-SARS-CoV-2 infection lymphocyte count and the presence of common co-morbidities increased the risk of mortality in PID. Access to specific COVID-19 treatments in this cohort was limited: only 22.9% (n = 33/144) of patients admitted to the hospital received dexamethasone, remdesivir, an anti-SARS-CoV-2 antibody-based therapeutic (e.g. REGN-COV2 or convalescent plasma) or tocilizumab as a monotherapy or in combination. Dexamethasone, remdesivir, and anti-SARS-CoV-2 antibody-based therapeutics appeared efficacious in PID and SID. Compared to the general population, individuals with PID or SID are at high risk of mortality following SARS-CoV-2 infection. Increasing age, low baseline lymphocyte count, and the presence of co-morbidities are additional risk factors for poor outcome in this cohort.

Dynamic clamping human and rabbit atrial calcium current: narrowing ICaL window abolishes early afterdepolarizations.

KEY POINTS: Early-afterdepolarizations (EADs) are abnormal action potential oscillations and a known cause of cardiac arrhythmias. Ventricular EADs involve reactivation of a Ca2+ current (ICaL ) in its 'window region' voltage range. However, electrical mechanisms of atrial EADs, a potential cause of atrial fibrillation, are poorly understood. Atrial cells were obtained from consenting patients undergoing heart surgery, as well as from rabbits. ICaL was blocked with nifedipine and then a hybrid patch clamp/mathematical-modelling technique, 'dynamic clamping', was used to record action potentials at the same time as injecting an artificial, modifiable, ICaL (ICaL,D-C ). Progressively widening the ICaL,D-C window region produced EADs of various types, dependent on window width. EAD production was strongest upon moving the activation (vs. inactivation) side of the window. EADs were then induced by a different method: increasing ICaL,D-C amplitude and/or K+ channel-blockade (4-aminopyridine). Narrowing of the ICaL,D-C window by ∼10 mV abolished these EADs. Atrial ICaL window narrowing is worthy of further testing as a potential anti-atrial fibrillation drug mechanism. ABSTRACT: Atrial early-afterdepolarizations (EADs) may contribute to atrial fibrillation (AF), perhaps involving reactivation of L-type Ca2+ current (ICaL ) in its window region voltage range. The present study aimed (i) to validate the dynamic clamp technique for modifying the ICaL contribution to atrial action potential (AP) waveform; (ii) to investigate the effects of widening the window ICaL on EAD-propensity; and (iii) to test whether EADs from increased ICaL and AP duration are supressed by narrowing the window ICaL . ICaL and APs were recorded from rabbit and human atrial myocytes by whole-cell-patch clamp. During AP recording, ICaL was inhibited (3 µm nifedipine) and replaced by a dynamic clamp model current, ICaL,D-C (tuned to native ICaL characteristics), computed in real-time (every 50 µs) based on myocyte membrane potential. ICaL,D-C -injection restored the nifedipine-suppressed AP plateau. Widening the window ICaL,D-C , symmetrically by stepwise simultaneous equal shifts of half-voltages (V0.5 ) of ICaL,D-C activation (negatively) and inactivation (positively), generated EADs (single, multiple or preceding repolarization failure) in a window width-dependent manner, as well as AP alternans. A stronger EAD-generating effect resulted from independently shifting activation V0.5 (asymmetrical widening) than inactivation V0.5 ; for example, a 15 mV activation shift produced EADs in nine of 17 (53%) human atrial myocytes vs. 0 of 18 from inactivation shift (P < 0.05). In 11 rabbit atrial myocytes in which EADs were generated either by increasing the conductance of normal window width ICaL,D-C or subsequent 4-aminopyridine (2 mm), window ICaL,D-C narrowing (10 mV) abolished EADs of all types (P < 0.05). The present study validated the dynamic clamp for ICaL , which is novel in atrial cardiomyocytes, and showed that EADs of various types are generated by widening (particularly asymmetrically) the window ICaL , as well as abolished by narrowing it. Window ICaL narrowing is a potential therapeutic mechanism worth pursuing in the search for improved anti-AF drugs.

Practicalities of a reduced volume formulation of a C1-INH concentrate for the treatment of hereditary angioedema: real-life experience.

BACKGROUND: Hereditary angioedema (HAE) due to C1 esterase inhibitor (C1-INH) deficiency is characterized by recurrent swelling attacks that can be life-threatening if left untreated. Prompt treatment is vital during acute attacks; plasma-derived C1-INH (Berinert®) is one treatment currently licensed for the intravenous treatment of acute HAE attacks in adults, adolescents and children. A new, volume-reduced formulation, of C1-INH is currently available which aims to reduce the time to treatment, and provide greater convenience to patients and healthcare professionals. Here we compare the clinical experience of the reduced volume 1500 IU vial with multiple 500 IU vials. METHODS: HAE patients treated with C1-INH at the Royal London Hospital were selected to take part in this assessment. Included patients were aged 10-65 with moderate to severe HAE requiring high doses of C1-INH. The practicalities of the reduced 1500 IU vial compared with multiple 500 IU vials were assessed, including preparation and administration time, training to self-administer time and several quality of life aspects. RESULTS: Twenty-three patients participated in this study. Twenty-one patients were previously treated with C1-INH (Berinert®) 500 IU for 1-14 years prior to switching to the 1500 IU vial format, two patients were naïve to C1-INH (Berinert®). Preparation and administration of C1-INH (Berinert®) 1500 IU was faster than an equivalent dose with multiple 500 IU vials (11 and 17 min, respectively) and also required less time to train to self-administer (45 and 55 min, respectively). Overall, patients rated the 1500 IU vial format higher in all assessed aspects than the 500 IU format, including preparation, administration, training, travel and storage. Nonetheless, reconstitution of the 1500 IU vial was noted more difficult, requiring gentle mixing to fully dissolve prior to intravenous injection. Patients remained stable on C1-INH (Berinert®) 1500 IU; two patients switched back to multiple 500 IU vials due to headaches and preference for a larger volume. CONCLUSIONS: The volume-reduced C1-INH concentrate (Berinert®) 1500 IU is a practical and convenient alternative to multiple 500 IU vials for the treatment of HAE, which provides patients with more control and independence over their disease owing to a simpler to administer treatment.

Fast Optical Sectioning for Widefield Fluorescence Mesoscopy with the Mesolens based on HiLo Microscopy.

We present here a fast optical sectioning method for mesoscopy based on HiLo microscopy, which makes possible imaging of specimens of up to 4.4 mm × 3 mm × 3 mm in volume in under 17 hours (estimated for a z-stack comprising 1000 images excluding computation time) with subcellular resolution throughout. Widefield epifluorescence imaging is performed with the Mesolens using a high pixel-number camera capable of sensor-shifting to generate a 259.5 Megapixel image, and we have developed custom software to perform HiLo processing of the very large datasets. Using this method, we obtain comparable sectioning strength to confocal laser scanning microscopy (CLSM), with sections as thin as 6.8 ± 0.2 µm and raw acquisition speed of 1 minute per slice which is up to 30 times faster than CLSM on the full field of view (FOV) of the Mesolens of 4.4 mm with lateral resolution of 0.7 µm and axial resolution of 7 µm. We have applied this HiLo mesoscopy method to image fixed and fluorescently stained hippocampal neuronal specimens and a 5-day old zebrafish larva.

Real-world outcomes in hereditary angioedema: first experience from the Icatibant Outcome Survey in the United Kingdom.

BACKGROUND: Hereditary angioedema (HAE) is a potentially life-threatening, bradykinin-mediated disease, often misdiagnosed and under-treated, with long diagnostic delays. There are limited real-world data on best-practice management of HAE in the UK. OBJECTIVES: To characterize the clinical profile, management and outcomes of patients with HAE type I and II from three specialist centres in the UK using data from the Icatibant Outcome Survey (IOS; Shire, Zug, Switzerland), an international observational study monitoring safety and effectiveness of icatibant, a selective bradykinin B2 receptor antagonist. METHODS: We performed retrospective analyses of IOS data for patients with HAE type I and II from three centres in the UK and compared UK data with pooled IOS data from 10 countries (48 centres). RESULTS: Analyses included 73 UK and 579 non-UK patients with HAE type I or II. Median diagnostic delay was 6.2 and 5.9 years, respectively. Analysis of data collected from February 2008 to July 2016 included 286 icatibant-treated attacks in 58 UK patients and 2553 icatibant-treated attacks in 436 non-UK patients (median of 3.0 attacks per patient in both groups). More attacks were treated by icatibant self-administration in UK patients (95.8%) than in non-UK patients (86.8%, p < 0.001). Time to icatibant treatment, time to resolution and attack duration were not significantly different in the UK versus non-UK patients. CONCLUSION: UK patients from the specialist centres studied report similar diagnostic delay and similar icatibant treatment outcomes to their non-UK counterparts. However, improvements in the timely diagnosis of HAE are still required.Trial registration ClinicalTrials.gov NCT01034969.

A novel optical microscope for imaging large embryos and tissue volumes with sub-cellular resolution throughout.

Current optical microscope objectives of low magnification have low numerical aperture and therefore have too little depth resolution and discrimination to perform well in confocal and nonlinear microscopy. This is a serious limitation in important areas, including the phenotypic screening of human genes in transgenic mice by study of embryos undergoing advanced organogenesis. We have built an optical lens system for 3D imaging of objects up to 6 mm wide and 3 mm thick with depth resolution of only a few microns instead of the tens of microns currently attained, allowing sub-cellular detail to be resolved throughout the volume. We present this lens, called the Mesolens, with performance data and images from biological specimens including confocal images of whole fixed and intact fluorescently-stained 12.5-day old mouse embryos.

Expert perspectives on hereditary angioedema: Key areas for advancements in care across the patient journey.

BACKGROUND: Published literature documents the substantial burden of hereditary angioedema (HAE) with C1 inhibitor deficiency on the quality of life and work productivity of patients. However, despite advances in the field and the availability of guidelines to advise health care providers (HCP) on the diagnosis and management of HAE, there are still many challenges to overcome. For example, delayed diagnosis and misdiagnosis are common, and treatment practices vary worldwide. OBJECTIVE: An international expert panel was convened to consider opportunities for improvements that would benefit patients with HAE. METHODS: Based on professional and personal experiences, the experts developed schematics to describe the journey of patients through the following stages: (1) onset of symptoms and initial evaluation; (2) referral/diagnosis; and (3) management of HAE. More importantly, the panel identified key areas in which it was possible to optimize the support provided to patients and HCPs along this journey. RESULTS: Overall, this approach highlighted the need for wider dissemination of algorithms and scientific data to more effectively educate HCPs from multiple disciplines and the need for more research to inform appropriate treatment decisions. Furthermore, HAE awareness campaigns, accurate online information, and referral to patient advocacy groups were all considered helpful approaches to support patients. CONCLUSION: More detailed and widespread information on the diagnosis and management of HAE is needed and may lead to advancements in care throughout the journey of the patient with HAE.

The transition of smooth muscle cells from a contractile to a migratory, phagocytic phenotype: direct demonstration of phenotypic modulation.

KEY POINTS: Smooth muscle cell (SMC) phenotypic conversion from a contractile to a migratory phenotype is proposed to underlie cardiovascular disease but its contribution to vascular remodelling and even its existence have recently been questioned. Tracking the fate of individual SMCs is difficult as no specific markers of migratory SMCs exist. This study used a novel, prolonged time-lapse imaging approach to continuously track the behaviour of unambiguously identified, fully differentiated SMCs. In response to serum, highly-elongated, contractile SMCs initially rounded up, before spreading and migrating and these migratory cells displayed clear phagocytic activity. This study provides a direct demonstration of the transition of fully contractile SMCs to a non-contractile, migratory phenotype with phagocytic capacity that may act as a macrophage-like cell. ABSTRACT: Atherosclerotic plaques are populated with smooth muscle cells (SMCs) and macrophages. SMCs are thought to accumulate in plaques because fully differentiated, contractile SMCs reprogramme into a 'synthetic' migratory phenotype, so-called phenotypic modulation, whilst plaque macrophages are thought to derive from blood-borne myeloid cells. Recently, these views have been challenged, with reports that SMC phenotypic modulation may not occur during vascular remodelling and that plaque macrophages may not be of haematopoietic origin. Following the fate of SMCs is complicated by the lack of specific markers for the migratory phenotype and direct demonstrations of phenotypic modulation are lacking. Therefore, we employed long-term, high-resolution, time-lapse microscopy to track the fate of unambiguously identified, fully-differentiated, contractile SMCs in response to the growth factors present in serum. Phenotypic modulation was clearly observed. The highly elongated, contractile SMCs initially rounded up, for 1-3 days, before spreading outwards. Once spread, the SMCs became motile and displayed dynamic cell-cell communication behaviours. Significantly, they also displayed clear evidence of phagocytic activity. This macrophage-like behaviour was confirmed by their internalisation of 1 µm fluorescent latex beads. However, migratory SMCs did not uptake acetylated low-density lipoprotein or express the classic macrophage marker CD68. These results directly demonstrate that SMCs may rapidly undergo phenotypic modulation and develop phagocytic capabilities. Resident SMCs may provide a potential source of macrophages in vascular remodelling.

Quantification of global mitochondrial DNA methylation levels and inverse correlation with age at two CpG sites.

Mammalian ageing features biological attrition evident at cellular, genetic and epigenetic levels. Mutation of mitochondrial DNA, and nuclear DNA methylation changes are well established correlates of ageing. The methylation of mitochondrial DNA (mtDNA) is a new and incompletely described phenomenon with unknown biological control and significance. Here we describe the bisulphite sequencing of mtDNA from 82 individuals aged 18-91 years. We detected low and variable levels of mtDNA methylation at 54 of 133 CpG sites interrogated. Regression analysis of methylation levels at two CpG sites (M1215 and M1313) located within the 12S ribosomal RNA gene showed an inverse correlation with subject age suggesting their utility as epigenetic markers of ageing.

Widefield Two-Photon Excitation without Scanning: Live Cell Microscopy with High Time Resolution and Low Photo-Bleaching.

We demonstrate fluorescence imaging by two-photon excitation without scanning in biological specimens as previously described by Hwang and co-workers, but with an increased field size and with framing rates of up to 100 Hz. During recordings of synaptically-driven Ca(2+) events in primary rat hippocampal neurone cultures loaded with the fluorescent Ca(2+) indicator Fluo-4 AM, we have observed greatly reduced photo-bleaching in comparison with single-photon excitation. This method, which requires no costly additions to the microscope, promises to be useful for work where high time-resolution is required.

Primary vs. secondary antibody deficiency: clinical features and infection outcomes of immunoglobulin replacement.

Secondary antibody deficiency can occur as a result of haematological malignancies or certain medications, but not much is known about the clinical and immunological features of this group of patients as a whole. Here we describe a cohort of 167 patients with primary or secondary antibody deficiencies on immunoglobulin (Ig)-replacement treatment. The demographics, causes of immunodeficiency, diagnostic delay, clinical and laboratory features, and infection frequency were analysed retrospectively. Chemotherapy for B cell lymphoma and the use of Rituximab, corticosteroids or immunosuppressive medications were the most common causes of secondary antibody deficiency in this cohort. There was no difference in diagnostic delay or bronchiectasis between primary and secondary antibody deficiency patients, and both groups experienced disorders associated with immune dysregulation. Secondary antibody deficiency patients had similar baseline levels of serum IgG, but higher IgM and IgA, and a higher frequency of switched memory B cells than primary antibody deficiency patients. Serious and non-serious infections before and after Ig-replacement were also compared in both groups. Although secondary antibody deficiency patients had more serious infections before initiation of Ig-replacement, treatment resulted in a significant reduction of serious and non-serious infections in both primary and secondary antibody deficiency patients. Patients with secondary antibody deficiency experience similar delays in diagnosis as primary antibody deficiency patients and can also benefit from immunoglobulin-replacement treatment.

Use of recombinant C1 inhibitor in patients with resistant or frequent attacks of hereditary or acquired angioedema.

BACKGROUND: Conestat alfa (Ruconest, rhC1INH) is the first recombinant human C1 inhibitor protein (C1INH) for the treatment of acute attacks of hereditary angioedema (HAE). OBJECTIVE: To assess clinical experience of the first 11 adult patients who received rhC1INH in clinical practice in the UK. METHODS: Eleven patients (nine HAE type 1, one HAE type 2 and one acquired angioedema with C1 inhibitor deficiency) received between one and six, mostly self-administered, doses of rhC1INH for acute HAE attacks. They were asked to record their time to first response and complete resolution following the treatment. This cohort included our most severely affected and difficult to treat patients. RESULTS: In most cases, time to first improvement following rhC1INH and complete resolution was recorded as comparable to their typical response to pdC1INH, although 4/11 patients reported that the time to first improvement was much quicker than their average pdC1INH response. Five of the 11 patients continued with rhC1INH as their preferred rescue treatment. Of those who chose not to continue the treatment, four reported a recurrence or early return of symptoms with rhC1INH. CONCLUSION: In our experience, rhC1INH is a beneficial treatment for patients with preference for a C1INH that is not plasma derived and it is suitable for home treatment. In some cases it demonstrates cost saving, especially for heavier patients who require higher doses. In some patients rhC1INH may result in faster resolution of symptoms. It may be associated with an early return of symptoms in patients with exceptionally frequent attacks.

Characterisation of Weibel-Palade body fusion by amperometry in endothelial cells reveals fusion pore dynamics and the effect of cholesterol on exocytosis.

Regulated secretion from endothelial cells is mediated by Weibel-Palade body (WPB) exocytosis. Plasma membrane cholesterol is implicated in regulating secretory granule exocytosis and fusion pore dynamics; however, its role in modulating WPB exocytosis is not clear. To address this we combined high-resolution electrochemical analysis of WPB fusion pore dynamics, by amperometry, with high-speed optical imaging of WPB exocytosis following cholesterol depletion or supplementation in human umbilical vein endothelial cells. We identified serotonin (5-HT) immunoreactivity in WPBs, and VMAT1 expression allowing detection of secreted 5-HT as discrete current spikes during exocytosis. A high proportion of spikes (∼75%) had pre-spike foot signals, indicating that WPB fusion proceeds via an initial narrow pore. Cholesterol depletion significantly reduced pre-spike foot signal duration and increased the rate of fusion pore expansion, whereas cholesterol supplementation had broadly the reverse effect. Cholesterol depletion slowed the onset of hormone-evoked WPB exocytosis, whereas its supplementation increased the rate of WPB exocytosis and hormone-evoked proregion secretion. Our results provide the first analysis of WPB fusion pore dynamics and highlight an important role for cholesterol in the regulation of WPB exocytosis.

Management of hereditary angioedema.

Hereditary angioedema is characterised by unpredictable, painful and potentially life-threatening oedema. Recently, some C1 inhibitors have been approved for self-administration and/or routine prevention, enabling patients to be proactive in managing their disease and reducing the burden of illness. This article discusses the effect of these advances from a specialist nurse's perspective.

Light-induced Ca(2+) transients observed in widefield epi-fluorescence microscopy of excitable cells.

We have investigated the possibility that variations in the level of intracellular Ca(2+) in excitable cells might be induced as an artifact of the incoherent illumination that is being used to monitor transient responses. In order to avoid the fluctuations in power of an arc lamp source, a microscope using a light emitting diode that was calibrated accurately at low power levels, was constructed to provide good control over the dose of light applied to the biological specimen. We report here that higher powers of illumination increased the probability of occurrence of Ca(2+) transients even in the sub-mW range normally used to measure such transients in epi-fluorescence work, suggesting that caution should be exercised when designing experiments and interpreting data.

A promising new wavelength region for three-photon fluorescence microscopy of live cells.

We report three-photon laser scanning microscopy (3PLSM) using a bi-directional pumped optical parametric oscillator (OPO) with signal wavelength output at λ= 1500 nm. This novel laser was used to overcome the high optical loss in the infrared spectral region observed in laser scanning microscopes and objective lenses that renders them otherwise difficult to use for imaging. To test our system, we performed 3PLSM auto-fluorescence imaging of live plant cells at λ= 1500 nm, specifically Spirogyra, and compared performance with two-photon excitation (2PLSM) imaging using a femtosecond pulsed Ti:Sapphire laser at λ= 780 nm. Analysis of cell viability based on cytoplasmic organelle streaming and structural changes of cells revealed that at similar peak powers, 2PLSM caused gross cell damage after 5 min but 3PLSM showed little or no interference with cell function after 15 min. The λ= 1500 nm OPO is thus shown to be a practical laser source for live cell imaging.

Remodelling of human atrial K+ currents but not ion channel expression by chronic ß-blockade.

Chronic ß-adrenoceptor antagonist (ß-blocker) treatment in patients is associated with a potentially anti-arrhythmic prolongation of the atrial action potential duration (APD), which may involve remodelling of repolarising K(+) currents. The aim of this study was to investigate the effects of chronic ß-blockade on transient outward, sustained and inward rectifier K(+) currents (I(TO), I(KSUS) and I(K1)) in human atrial myocytes and on the expression of underlying ion channel subunits. Ion currents were recorded from human right atrial isolated myocytes using the whole-cell-patch clamp technique. Tissue mRNA and protein levels were measured using real time RT-PCR and Western blotting. Chronic ß-blockade was associated with a 41% reduction in I(TO) density: 9.3 ± 0.8 (30 myocytes, 15 patients) vs 15.7 ± 1.1 pA/pF (32, 14), p < 0.05; without affecting its voltage-, time- or rate dependence. I(K1) was reduced by 34% at -120 mV (p < 0.05). Neither I(KSUS), nor its increase by acute ß-stimulation with isoprenaline, was affected by chronic ß-blockade. Mathematical modelling suggested that the combination of I(TO)- and I(K1)-decrease could result in a 28% increase in APD(90). Chronic ß-blockade did not alter mRNA or protein expression of the I(TO) pore-forming subunit, Kv4.3, or mRNA expression of the accessory subunits KChIP2, KChAP, Kvß1, Kvß2 or frequenin. There was no reduction in mRNA expression of Kir2.1 or TWIK to account for the reduction in I(K1). A reduction in atrial I(TO) and I(K1) associated with chronic ß-blocker treatment in patients may contribute to the associated action potential prolongation, and this cannot be explained by a reduction in expression of associated ion channel subunits.