Regulation of A disintegrin and metalloproteinase (ADAM) family sheddases ADAM10 and ADAM17: The emerging role of tetraspanins and rhomboids

A disintegrin and metalloprotease (ADAM) 10 and ADAM17 are ubiquitous transmembrane “molecular scissors” which proteolytically cleave, or shed, the extracellular regions of other transmembrane proteins. ADAM10 is essential for development because it cleaves Notch proteins to induce Notch signaling and regulate cell fate decisions. ADAM17 is regarded as a first line of defense against injury and infection, by releasing tumor necrosis factor α (TNFα) to promote inflammation and epidermal growth factor (EGF) receptor ligands to maintain epidermal barrier function. However, the regulation of ADAM10 and ADAM17 trafficking and activation are not fully understood. This review will describe how the TspanC8 subgroup of tetraspanins (Tspan5, 10, 14, 15, 17, and 33) and the iRhom subgroup of protease-inactive rhomboids (iRhom1 and 2) have emerged as important regulators of ADAM10 and ADAM17, respectively. In particular, they are required for the enzymatic maturation and trafficking to the cell surface of the ADAMs, and there is evidence that different TspanC8s and iRhoms target the ADAMs to distinct substrates. The TspanC8s and iRhoms have not been studied functionally on platelets. On these cells, ADAM10 is the principal sheddase for the platelet collagen receptor GPVI, and the regulatory TspanC8s are Tspan14, 15, and 33, as determined from proteomic data. Platelet ADAM17 is the sheddase for the von Willebrand factor (vWF) receptor GPIb, and iRhom2 is the only iRhom that is expressed. Induced shedding of either GPVI or GPIb has therapeutic potential, since inhibition of either receptor is regarded as a promising anti-thrombotic therapy. Targeting of Tspan14, 15, or 33 to activate platelet ADAM10, or iRhom2 to activate ADAM17, may enable such an approach to be realized, without the toxic side effects of activating the ADAMs on every cell in the body

Familial atrial fibrillation mutation M1875T-SCN5A increases early sodium current and dampens the effect of flecainide

Aims Atrial fibrillation (AF) is the most common cardiac arrhythmia. Pathogenic variants in genes encoding ion channels are associated with familial AF. The point mutation M1875T in the SCN5A gene, which encodes the α-subunit of the cardiac sodium channel Nav1.5, has been associated with increased atrial excitability and familial AF in patients. Methods and results We designed a new murine model carrying the Scn5a-M1875T mutation enabling us to study the effects of the Nav1.5 mutation in detail in vivo and in vitro using patch clamp and microelectrode recording of atrial cardiomyocytes, optical mapping, electrocardiogram, echocardiography, gravimetry, histology, and biochemistry. Atrial cardiomyocytes from newly generated adult Scn5a-M1875T+/− mice showed a selective increase in the early (peak) cardiac sodium current, larger action potential amplitude, and a faster peak upstroke velocity. Conduction slowing caused by the sodium channel blocker flecainide was less pronounced in Scn5a-M1875T+/− compared to wildtype atria. Overt hypertrophy or heart failure in Scn5a-M1875T+/− mice could be excluded. Conclusion The Scn5a-M1875T point mutation causes gain-of-function of the cardiac sodium channel. Our results suggest increased atrial peak sodium current as a potential trigger for increased atrial excitability

Insights into the Role of a Cardiomyopathy-Causing Genetic Variant in ACTN2

Pathogenic variants in ACTN2, coding for alpha-actinin 2, are known to be rare causes of Hyper-trophic Cardiomyopathy. However, little is known about the underlying disease mechanisms. Adult heterozygous mice carrying the Actn2 p.Met228Thr variant were phenotyped by echocar-diography. For homozygous mice, viable E15.5 embryonic hearts were analysed by High Reso-lution Episcopic Microscopy and wholemount staining, complemented by unbiased proteomics, qPCR and Western blotting. Heterozygous Actn2 p.Met228Thr mice have no overt phenotype. Only mature males show molecular parameters indicative of cardiomyopathy. By contrast, the variant is embryonically lethal in the homozygous setting and E15.5 hearts show multiple morphological abnormalities. Molecular analyses, including unbiased proteomics, identified quantitative abnormalities in sarcomeric parameters, cell cycle defects and mitochondrial dys-function. The mutant alpha-actinin protein is found to be destabilised, associated with increased activity of the ubiquitin-proteosomal system. This missense variant in alpha-actinin renders the protein less stable. In response, the ubiquitin-proteosomal system is activated; a mechanism which has been implicated in cardiomyopathies previously. In parallel, lack of functional al-pha-actinin is thought to cause energetic defects through mitochondrial dysfunction. This seems, together with cell cycle defects, the likely cause of death of the embryos. The defects also have wide-ranging morphological consequences

An angiopoietin 2, FGF23, and BMP10 biomarker signature differentiates atrial fibrillation from other concomitant cardiovascular conditions

Abstract Early detection of atrial fibrillation (AF) enables initiation of anticoagulation and early rhythm control therapy to reduce stroke, cardiovascular death, and heart failure. In a cross-sectional, observational study, we aimed to identify a combination of circulating biomolecules reflecting different biological processes to detect prevalent AF in patients with cardiovascular conditions presenting to hospital. Twelve biomarkers identified by reviewing literature and patents were quantified on a high-precision, high-throughput platform in 1485 consecutive patients with cardiovascular conditions (median age 69 years [Q1, Q3 60, 78]; 60% male). Patients had either known AF (45%) or AF ruled out by 7-day ECG-monitoring. Logistic regression with backward elimination and a neural network approach considering 7 key clinical characteristics and 12 biomarker concentrations were applied to a randomly sampled discovery cohort (n = 933) and validated in the remaining patients (n = 552). In addition to age, sex, and body mass index (BMI), BMP10, ANGPT2, and FGF23 identified patients with prevalent AF (AUC 0.743 [95% CI 0.712, 0.775]). These circulating biomolecules represent distinct pathways associated with atrial cardiomyopathy and AF. Neural networks identified the same variables as the regression-based approach. The validation using regression yielded an AUC of 0.719 (95% CI 0.677, 0.762), corroborated using deep neural networks (AUC 0.784 [95% CI 0.745, 0.822]). Age, sex, BMI and three circulating biomolecules (BMP10, ANGPT2, FGF23) are associated with prevalent AF in unselected patients presenting to hospital. Findings should be externally validated. Results suggest that age and different disease processes approximated by these three biomolecules contribute to AF in patients. Our findings have the potential to improve screening programs for AF after external validation

Chronic activation of human cardiac fibroblasts in vitro attenuates the reversibility of the myofibroblast phenotype

Abstract Activation of cardiac fibroblasts and differentiation to myofibroblasts underlies development of pathological cardiac fibrosis, leading to arrhythmias and heart failure. Myofibroblasts are characterised by increased α-smooth muscle actin (α-SMA) fibre expression, secretion of collagens and changes in proliferation. Transforming growth factor-beta (TGF-β) and increased mechanical stress can initiate myofibroblast activation. Reversibility of the myofibroblast phenotype has been observed in murine cells but has not been explored in human cardiac fibroblasts. In this study, chronically activated adult primary human ventricular cardiac fibroblasts and human induced pluripotent stem cell derived cFbs (hiPSC-cFbs) were used to investigate the potential for reversal of the myofibroblast phenotype using either subculture on soft substrates or TGF-β receptor inhibition. Culture on softer plates (25 or 2 kPa Young’s modulus) did not alter proliferation or reduce expression of α-SMA and collagen 1. Similarly, culture of myofibroblasts in the presence of TGF-β inhibitor did not reverse myofibroblasts back to a quiescent phenotype. Chronically activated hiPSC-cFbs also showed attenuated response to TGF-β receptor inhibition and inability to reverse to quiescent fibroblast phenotype. Our data demonstrate substantial loss of TGF-β signalling plasticity as well as a loss of feedback from the surrounding mechanical environment in chronically activated human myofibroblasts

Poster Session B

Introduction Atrial fibrillation (AF) affects over 1% of the population and is a leading cause of stroke and heart failure in the elderly. Sodium channel blockade is a common treatment for AF. A feared side effect of inhibitors of the cardiac voltage-gated sodium current (INa), such a flecainide, however, is ventricular arrhythmia. We investigated the biophysical reasons for the relative safety of sodium channel blockers. Methods Whole cell patch clamping was performed to measure INa and action potentials (APs). Optical mapping of the LA and LV was performed in the intact mouse heart using voltage dye Di-4- ANEPPS and analysed using ElectroMap (1). LA and LV INa data were used to model changes in AP morphology using a modified mathematical model of the ventricular cardiomyocyte (2). Expression of NaV1.5, NaVβ2 and NaVβ4 was measured by Western blotting and SCN5A, SCN4B and SCN2B by RNAseq analysis in matched mouse and non-failing human LA and LV. Results At physiological holding potentials (-75mV), LA peak INa current density was significantly reduced compared to LV. Western blotting data demonstrate that this is not due to reduced atrial NaV1.5/SCN5A expression in either mouse or human. AP recordings revealed LA cells exhibit reduced upstroke velocity (238.6±27.7vs304.2±27.9mV/ms, p=0.0018) compared to the LV (n=23-40/5 cells/mice). Modelling studies confirmed these findings. At all holding potentials, 1µM flecainide inhibited INa to a larger extent in the LA compared to LV cells (Figure 1). Furthermore, flecainide decreased AP upstroke velocity to a higher degree in LA (47.9±18.8%) when compared to LV cells (18.6±9.8%, p=0.04, n=4 mice). Optical mapping demonstrated reduced conduction velocity in the LA (24.4±2.7cm/s) compared to LV (36.1±6.2cm/s, p=0.03, n=5). Flecainide significantly decreased conduction velocity in the LA (-40.4±7.2%, p Conclusion Significant differences exist in the biophysical properties of sodium channels in the LA and LV, and their response to flecainide. The reduced effectiveness of flecainide in the LV can explain the relative safety of sodium channel blocker therapy