Nicola Hellen

Action Potential Morphology of Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes Does Not Predict Cardiac Chamber Specificity and Is Dependent on Cell Density

Previous studies investigating human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) have proposed the distinction of heart chamber-specific (atrial, ventricular, pacemaker) electrophysiological phenotypes based on action potential (AP) morphology. This suggestion has been based on data acquired using techniques that allow measurements from only a small number of cells and at low seeding densities. It has also been observed that density of culture affects the properties of iPSC-CMs. Here we systematically analyze AP morphology from iPSC-CMs at two seeding densities: 60,000 cells/well (confluent monolayer) and 15,000 cells/well (sparsely-seeded) using a noninvasive optical method. The confluent cells (n = 360) demonstrate a series of AP morphologies on a normally distributed spectrum with no evidence for specific subpopulations. The AP morphologies of sparsely seeded cells (n = 32) displayed a significantly different distribution, but even in this case there is no clear evidence of chamber-specificity. Reduction in gap junction conductance using carbenoxolone only minimally affected APD distribution in confluent cells. These data suggest that iPSC-CMs possess a sui generis AP morphology, and when observed in different seeding densities may encompass any shape including those resembling chamber-specific subtypes. These results may be explained by different functional maturation due to culture conditions.

STXBP1 promotes Weibel-Palade body exocytosis through its interaction with the Rab27A effector Slp4-a

Vascular endothelial cells contain unique rod-shaped secretory organelles, called Weibel-Palade bodies (WPBs), which contain the hemostatic protein von Willebrand factor (VWF) and a cocktail of angiogenic and inflammatory mediators. We have shown that the Rab27A effector synaptotagmin-like protein 4-a (Slp4-a) plays a critical role in regulating hormone-evoked WPB exocytosis. Using a nonbiased proteomic screen for targets for Slp4-a, we now identify syntaxin-binding protein 1 (STXBP1) and syntaxin-2 and -3 as endogenous Slp4-a binding partners in endothelial cells. Coimmunoprecipitations showed that STXBP1 interacts with syntaxin-2 and -3, but not with syntaxin-4. Small interfering RNA-mediated silencing of STXBP1 expression impaired histamine- and forskolin-induced VWF secretion. To further substantiate the role of STXBP1, we isolated blood outgrowth endothelial cells (BOECs) from an early infantile epileptic encephalopathy type 4 (EIEE4) patient carrying a de novo mutation in STXBP1. STXBP1-haploinsufficient EIEE4 BOECs contained similar numbers of morphologically normal WPBs compared with control BOECs of healthy donors; however, EIEE4 BOECs displayed significantly impaired histamine- and forskolin-stimulated VWF secretion. Based on these findings, we propose that the Rab27A-Slp4-a complex on WPB promotes exocytosis through an interaction with STXBP1, thereby controlling the release of vaso-active substances in the vasculature.

Protein mobilities and P-selectin storage in Weibel–Palade bodies

Using fluorescence recovery after photobleaching (FRAP) we measured the mobilities of EGFP-tagged soluble secretory proteins in the endoplasmic reticulum (ER) and in individual Weibel–Palade bodies (WPBs) at early (immature) and late (mature) stages in their biogenesis. Membrane proteins (P-selectin, CD63, Rab27a) were also studied in individual WPBs. In the ER, soluble secretory proteins were mobile; however, following insertion into immature WPBs larger molecules (VWF, Proregion, tPA) and P-selectin became immobilised, whereas small proteins (ssEGFP, eotaxin-3) became less mobile. WPB maturation led to further decreases in mobility of small proteins and CD63. Acute alkalinisation of mature WPBs selectively increased the mobilities of small soluble proteins without affecting larger molecules and the membrane proteins. Disruption of the Proregion–VWF paracrystalline core by prolonged incubation with NH4Cl rendered P-selectin mobile while VWF remained immobile. FRAP of P-selectin mutants revealed that immobilisation most probably involves steric entrapment of the P-selectin extracellular domain by the Proregion–VWF paracrystal. Significantly, immobilisation contributed to the enrichment of P-selectin in WPBs; a mutation of P-selectin preventing immobilisation led to a failure of enrichment. Together these data shed new light on the transitions that occur for soluble and membrane proteins following their entry and storage into post-Golgi-regulated secretory organelles.

The Fallacy of Assigning Chamber Specificity to iPSC Cardiac Myocytes from Action Potential Morphology

The production and identification of chamber-specific, induced pluripotent stem cell-derived cardiac myocytes (iPSC-CMs) is a crucial step in the application of this technology in physiology, pharmacology, and regenerative studies. The significance of toxicology tests would be strongly diminished if, for instance, drugs for atrial fibrillation were tested in ventricular myocytes or pathophysiological mechanisms involved in hypertrophic cardiomyopathy were studied in atrial myocytes. One could predict that the transplantation of nodal myocytes in the infarcted ventricle would have a strong arrhythmogenic potential and could be disastrous in the setting of myocardial repair strategies.

Interaction between MyRIP and the actin cytoskeleton regulates Weibel-Palade body trafficking and exocytosis

ABSTRACT Weibel–Palade body (WPB)–actin interactions are essential for the trafficking and secretion of von Willebrand factor; however, the molecular basis for this interaction remains poorly defined. Myosin Va (MyoVa or MYO5A) is recruited to WPBs by a Rab27A–MyRIP complex and is thought to be the prime mediator of actin binding, but direct MyRIP–actin interactions can also occur. To evaluate the specific contribution of MyRIP–actin and MyRIP–MyoVa binding in WPB trafficking and Ca2+-driven exocytosis, we used EGFP–MyRIP point mutants with disrupted MyoVa and/or actin binding and high-speed live-cell fluorescence microscopy. We now show that the ability of MyRIP to restrict WPB movement depends upon its actin-binding rather than its MyoVa-binding properties. We also show that, although the role of MyRIP in Ca2+-driven exocytosis requires both MyoVa- and actin-binding potential, it is the latter that plays a dominant role. In view of these results and together with the analysis of actin disruption or stabilisation experiments, we propose that the role of MyRIP in regulating WPB trafficking and exocytosis is mediated largely through its interaction with actin rather than with MyoVa. Summary: The role of MyRIP in restricting the movement and exocytosis of Weibel–Palade bodies in endothelial cells is mediated primarily through its actin- rather than myosin-Va-binding properties.

G-protein Coupled Receptor Signaling in Pluripotent Stem Cell-derived Cardiovascular Cells: Implications for Disease Modeling.

Human pluripotent stem cell derivatives show promise as an in vitro platform to study a range of human cardiovascular diseases. A better understanding of the biology of stem cells and their cardiovascular derivatives will help to understand the strengths and limitations of this new model system. G-protein coupled receptors (GPCRs) are key regulators of stem cell maintenance and differentiation and have an important role in cardiovascular cell signaling. In this review, we will therefore describe the state of knowledge concerning the regulatory role of GPCRs in both the generation and function of pluripotent stem cell derived-cardiomyocytes, -endothelial, and -vascular smooth muscle cells. We will consider how far the in vitro disease models recapitulate authentic GPCR signaling and provide a useful basis for discovery of disease mechanisms or design of therapeutic strategies.

Is there more than one way to unpack a Weibel-Palade body?

To the editor: Endothelial cells respond to vascular damage by secreting concatemers of the adhesive glycoprotein von Willebrand factor (VWF) to capture blood platelets and promote hemostasis. VWF is contained in large rod-shaped secretory granules called Weibel-Palade bodies (WPBs), and how VWF is stored in and released from these organelles is of considerable interest.1 Recently, a novel mechanism was described for VWF release in which an actomyosin ring forms around the WPB several seconds after its fusion with the plasma membrane to squeeze VWF from the WPB.2 This new mechanism was described in experiments using the potent secretagogue phorbol 12-myristate 13-acetate (PMA), and has received considerable attention.3-5 However, the mechanism of action of PMA differs in several key respects from that of physiological secretagogues, such as histamine, that elevate intracellular free calcium ion concentrations ([Ca2+]i). PMA action is characterized by a slow onset (tens of seconds to minutes) but a protracted (hours) period of WPB fusion that occurs without an increase in [Ca2+]i. VWF is released slowly from the WPB after fusion with the plasma membrane (tens of seconds), and secretion is prevented by inhibition of protein kinase C,6 myosin IIB (MyoIIB),2 or actin disruption or stabilization.7 In contrast, histamine (or ionomycin) triggers a rapid (<1 second) but transient (10-30 seconds) burst in WPB exocytosis.8 Ca2+-mediated VWF secretion is not blocked by protein kinase C inhibition6 or actin disruption,9 and early optical studies indicated that the initial expulsion of VWF occurs on a subsecond time scale (see Erent et al8 and references therein). On the basis of these observations we asked whether the actomyosin process represents a general mechanism for VWF release from WPBs. Our new data suggest not. First, live-cell imaging of fluorescent VWF–enhanced green fluorescent protein (VWF-EGFP) or VWF-propeptide-EGFP (VWFpp-EGFP) expulsion from individual WPBs shows this process to be fast (Figure 1Ai-ii). Second, dual-color imaging of endothelial cells coexpressing VWFpp-EGFP or VWFpp-mCherry and either TagRFP-actin (Figure 1Bi-ii) or MyoIIB-GFP (Figure 1Ci-ii) revealed no evidence of redistribution or accumulation of actin or MyoIIB to WPBs undergoing exocytosis. Third, MyoIIB inhibition by blebbistatin did not prevent histamine-evoked VWF secretion (Figure 1D), and we have reconfirmed that actin disruption or stabilization fails to prevent Ca2+-mediated VWF secretion. Because myosin II may subtly regulate the opening of secretory granule fusion pores (reviewed in Porat-Shliom et al3), we also performed amperometry studies but found no major effects of MyoIIB inhibition on WPB fusion pore formation or expansion (Figure 1E). Together, the data suggest that expulsion of VWF from WPBs during Ca2+-driven WPB exocytosis does not involve actomyosin. Figure 1 Fast actomyosin-independent VWF expulsion from WPBs during Ca2+-mediated exocytosis. In the absence of flow, secreted VWF concatemers form irregular-shaped patches on the cell surface and disperse slowly into solution. Importantly, the initial expulsion ... What other mechanism might account for fast actomyosin-independent VWF expulsion during Ca2+-mediated exocytosis? Studies of mucins, large multimeric glycoproteins closely related to VWF, suggest that the subsecond expulsion of these charged polymers from mucin granules is driven by ionic fluxes and water entry (discussed in Erent et al8). VWF, like mucins, is stored at high concentration, and the condensation and aggregation of these proteins are facilitated by charge shielding by cationic species including hydrogen ion (H+) and Ca2+. The acidic lumen of the WPB is particularly important for VWF expulsion. Loss of H+ following fusion pore formation precedes postfusion changes in WPB morphology and rapid VWF expulsion, and both processes can be blocked simply by lowering the external pH close to that of the prefusion mature WPB.10 Thus, VWF expulsion from WPBs during histamine- or Ca2+-mediated exocytosis utilizes a beautifully simple mechanism that depends, in essence, on the chemistry of VWF and the intracellular processes that ensure its condensation and aggregation for storage at high concentration. Is this mechanism likely to be of physiological relevance? Following injury, endothelial cells must function rapidly to minimize blood loss. The earliest event within these endothelial cells will be an increase in [Ca2+]i produced either by cell damage, physicomechanical stimulation, or acute activation by physiological mediators generated locally at the injury site (eg, thrombin, histamine, fibrin, adenine nucleotides, and peptidoleukotrienes). Together, these mediators ensure rapid VWF expulsion to the endothelial cell surface to capture platelets and facilitate hemostasis. Clearly, there is more than one way to unpack a WPB. Further studies will be needed to clarify the specific physiological conditions subserved by actomyosin-dependent and -independent VWF delivery to the endothelial cell surface.

Induced pluripotent stem cell modelling of HLHS underlines the contribution of dysfunctional NOTCH signalling to impaired cardiogenesis

Abstract Hypoplastic left heart syndrome (HLHS) is among the most severe forms of congenital heart disease. Although the consensus view is that reduced flow through the left heart during development is a key factor in the development of the condition, the molecular mechanisms leading to hypoplasia of left heart structures are unknown. We have generated induced pluripotent stem cells (iPSC) from five HLHS patients and two unaffected controls, differentiated these to cardiomyocytes and identified reproducible in vitro cellular and functional correlates of the HLHS phenotype. Our data indicate that HLHS-iPSC have a reduced ability to give rise to mesodermal, cardiac progenitors and mature cardiomyocytes and an enhanced ability to differentiate to smooth muscle cells. HLHS-iPSC-derived cardiomyocytes are characterised by a lower beating rate, disorganised sarcomeres and sarcoplasmic reticulum and a blunted response to isoprenaline. Whole exome sequencing of HLHS fibroblasts identified deleterious variants in NOTCH receptors and other genes involved in the NOTCH signalling pathway. Our data indicate that the expression of NOTCH receptors was significantly downregulated in HLHS-iPSC-derived cardiomyocytes alongside NOTCH target genes confirming downregulation of NOTCH signalling activity. Activation of NOTCH signalling via addition of Jagged peptide ligand during the differentiation of HLHS-iPSC restored their cardiomyocyte differentiation capacity and beating rate and suppressed the smooth muscle cell formation. Together, our data provide firm evidence for involvement of NOTCH signalling in HLHS pathogenesis, reveal novel genetic insights important for HLHS pathology and shed new insights into the role of this pathway during human cardiac development.

Meeting abstracts from the 64th British Thyroid Association Annual Meeting

• Users may freely distribute the URL that is used to identify this publication. • Users may download and/or print one copy of the publication from the University of Birmingham research portal for the purpose of private study or non-commercial research. • User may use extracts from the document in line with the concept of ‘fair dealing’ under the Copyright, Designs and Patents Act 1988 (?) • Users may not further distribute the material nor use it for the purposes of commercial gain.Graves’ orbitopathy (GO) is the main extrathyroidal manifestation of Graves’ disease. When fully expressed, it is characterized by inflammatory soft tissue changes, exophthalmos, ocular dysmotility causing diplopia, and, rarely, sight-threatening dysthyroid optic neuropathy (DON). The prevalence of GO among Graves’ patients seems lately declining, probably due to early diagnosis, early intervention on risk factors associated with its occurrence or progression (smoking, uncontrolled thyroid dysfunction), early correction of hyper and hypothyroidism. Only about 25–30% of newly diagnosed Graves’ hyperthyroids are affected with GO, which is usually mild and rarely progressive. Assessment of activity and severity of GO according to standardized criteria is fundamental to plan management. The European Thyroid Association and the European Group on Graves’ Orbitopathy (EUGOGO) have recently published the first guideline on management of GO. Mild GO usually requires only a watchful strategy, in addition to local measures (eye drops, ointments) and removal of risk factors. Intravenous glucocorticoids (ivGCs) are the first-line treatment for moderate-to-severe and active GO, as demonstrated by randomized clinical trials. When ivGCs fail or GO recurs after treatment withdrawal, options include a second course of ivGCs, oral GCs combined with orbital radiotherapy or cyclosporine, rituximab. Evidence that the any of the above treatment be effective in the context of a poor response to a first course of ivGCs is limited and should be investigated in larger studies. In addition to rituximab, ongoing investigations are exploring the role of other biologics targeting, e.g., the IGF-1 receptor or the IL-6 receptor, and results will probably available in 1–2 years. When GO has been treated medically and is inactive, rehabilitative surgery (orbital decompression, squint surgery, eyelid surgery) is often needed.

Meeting abstracts from the 64th British Thyroid Association Annual Meeting: Newcastle, UK. 13/05/2016

• Users may freely distribute the URL that is used to identify this publication. • Users may download and/or print one copy of the publication from the University of Birmingham research portal for the purpose of private study or non-commercial research. • User may use extracts from the document in line with the concept of ‘fair dealing’ under the Copyright, Designs and Patents Act 1988 (?) • Users may not further distribute the material nor use it for the purposes of commercial gain.Graves’ orbitopathy (GO) is the main extrathyroidal manifestation of Graves’ disease. When fully expressed, it is characterized by inflammatory soft tissue changes, exophthalmos, ocular dysmotility causing diplopia, and, rarely, sight-threatening dysthyroid optic neuropathy (DON). The prevalence of GO among Graves’ patients seems lately declining, probably due to early diagnosis, early intervention on risk factors associated with its occurrence or progression (smoking, uncontrolled thyroid dysfunction), early correction of hyper and hypothyroidism. Only about 25–30% of newly diagnosed Graves’ hyperthyroids are affected with GO, which is usually mild and rarely progressive. Assessment of activity and severity of GO according to standardized criteria is fundamental to plan management. The European Thyroid Association and the European Group on Graves’ Orbitopathy (EUGOGO) have recently published the first guideline on management of GO. Mild GO usually requires only a watchful strategy, in addition to local measures (eye drops, ointments) and removal of risk factors. Intravenous glucocorticoids (ivGCs) are the first-line treatment for moderate-to-severe and active GO, as demonstrated by randomized clinical trials. When ivGCs fail or GO recurs after treatment withdrawal, options include a second course of ivGCs, oral GCs combined with orbital radiotherapy or cyclosporine, rituximab. Evidence that the any of the above treatment be effective in the context of a poor response to a first course of ivGCs is limited and should be investigated in larger studies. In addition to rituximab, ongoing investigations are exploring the role of other biologics targeting, e.g., the IGF-1 receptor or the IL-6 receptor, and results will probably available in 1–2 years. When GO has been treated medically and is inactive, rehabilitative surgery (orbital decompression, squint surgery, eyelid surgery) is often needed.