Thomas Brand

Background: Congenital junctional ectopic tachycardia (cJET) is a rare, potentially life-threatening arrhythmia suspicious for a genetic basis, yet its molecular underpinnings remain incompletely defined. The POPDC2 gene, involved in cardiac pacemaking and membrane trafficking of interacting ion channels, has not previously been conclusively linked to human tachyarrhythmias. This study investigates a novel POPDC2 variant (p.Leu245Pro) identified in a family with autosomal dominant cJET. Methods: Exome sequencing was performed to identify co-segregating variants in the affected family. Functional analysis of the POPDC2 p.Leu245Pro variant was conducted by molecular dynamics (MD) simulations, a membrane targeting assay, and a bimolecular fluorescence complementation assay. Additionally, the impact of the variant on Nav1.5 and TREK-1 currents was characterized in Xenopus oocytes. Results: The p.Leu245Pro POPDC2 variant showed a destabilization of the POPDC1-POPDC2 dimer interface, resulting in impaired heterodimer formation and membrane localization. Electrophysiological studies in Xenopus oocytes demonstrated that the mutant protein significantly affected Nav1.5 and TREK-1 currents. These findings support a functional impact of the POPDC2 p.Leu245Pro variant relevant to cardiac conduction. Conclusions: Our results provide the first functional evidence implicating POPDC2 in cJET and support its role as a novel candidate gene in tachyarrhythmic disease. This study enhances the understanding of genetic contributions to cJET and suggests further investigation of POPDC2 in other forms of supraventricular tachyarrhythmias.

Purpose of Review: The association of left-right (LR) axis development with congenital heart disease (CHD). Recent Findings: CHD affects up to 1.2% of the population, with 3–7% of cases attributed to abnormal LR patterning. Despite this, disruption of LR axis development remains poorly understood. Here, we examine the molecular mechanisms of embryonic LR axis formation. We review novel findings about LR asymmetry genes, the role of cilia and the nodal flow. We debate whether flow-sensing is based on morphogens or involves mechanosensing and explore mechanisms involved in asymmetric morphogenesis of the heart, including the NODAL-PITX2 cassette, intrinsic cellular chirality, and cardiac neural crest cells. Finally, we discuss environmental factors affecting LR axis formation and the potential development of therapeutics. Summary: Understanding the genetic underpinnings of LR axis development and its effects on CHD is crucial for risk stratification of patients, leading to more effective treatment.

Popeye domain-containing (POPDC) proteins selectively bind cAMP and mediate cellular responses to sympathetic nervous system (SNS) stimulation. The first discovered human genetic variant (POPDC1^S201F) is associated with atrioventricular (AV) block, which is exacerbated by increased SNS activity. Zebrafish carrying the homologous mutation (popdc1^S191F) display a similar phenotype to humans. To investigate the impact of POPDC1 dysfunction on cardiac electrophysiology and intracellular calcium handling, homozygous popdc1^S191F and popdc1 knock-out (popdc1^KO) zebrafish larvae and adult isolated popdc1^S191F hearts were studied by functional fluorescent analysis. It was found that in popdc1^S191F and popdc1^KO larvae, heart rate (HR), AV delay, action potential (AP) and calcium transient (CaT) upstroke speed, and AP duration were less than in wild-type larvae, whereas CaT duration was greater. SNS stress by β-adrenergic receptor stimulation with isoproterenol increased HR, lengthened AV delay, slowed AP and CaT upstroke speed, and shortened AP and CaT duration, yet did not result in arrhythmias. In adult popdc1^S191F zebrafish hearts, there was a higher incidence of AV block, slower AP upstroke speed, and longer AP duration compared to wild-type hearts, with no differences in CaT. SNS stress increased AV delay and led to further AV block in popdc1^S191F hearts while decreasing AP and CaT duration. Overall, we have revealed that arrhythmogenic effects of POPDC1 dysfunction on cardiac electrophysiology and intracellular calcium handling in zebrafish are varied, but already present in early development, and that AV node dysfunction may underlie SNS-induced arrhythmogenesis associated with popdc1 mutation in adults.

Background: Beta-2 adrenergic receptors (β ₂ ARs) but not beta-2 adrenergic receptors (β ₁ ARs) form a functional complex with L-type Ca ²⁺ channels (LTCCs) on the cardiomyocyte membrane. However, how microdomain localization in the plasma membrane affects the function of these complexes is unknown. We aim to study the coupling between LTCC and β adrenergic receptors in different cardiomyocyte microdomains, the distinct involvement of PKA and CAMKII (Ca ²⁺ /calmodulin-dependent protein kinase II) and explore how this functional complex is disrupted in heart failure. Methods: Global signaling between LTCCs and β adrenergic receptors was assessed with whole-cell current recordings and western blot analysis. Super-resolution scanning patch-clamp was used to explore the local coupling between single LTCCs and β ₁ AR or β ₂ AR in different membrane microdomains in control and failing cardiomyocytes. Results: LTCC open probability (Po) showed an increase from 0.054±0.003 to 0.092±0.008 when β ₂ AR was locally stimulated in the proximity of the channel (2 AR was lost. Interestingly, local stimulation of β ₁ AR did not elicit any change in the Po of LTCCs, indicating a lack of proximal functional interaction between the two, but we confirmed a general activation of LTCC via β ₁ AR. By using blockers of PKA and CaMKII and a Caveolin-3-knockout mouse model, we conclude that the β ₂ AR-LTCC regulation requires the presence of caveolin-3 and the activation of the CaMKII pathway. By contrast, at a cellular “global” level PKA plays a major role downstream β ₁ AR and results in an increase in LTCC current. Conclusions: Regulation of the LTCC activity by proximity coupling mechanisms occurs only via β ₂ AR, but not β ₁ AR. This may explain how β ₂ ARs tune the response of LTCCs to adrenergic stimulation in healthy conditions. This coupling is lost in heart failure; restoring it could improve the adrenergic response of failing cardiomyocytes.

The Popeye domain containing (POPDC) genes encode sarcolemma-localized cAMP effector proteins. Mutations in blood vessel epicardial substance (BVES) also known as POPDC1 and POPDC2 have been associated with limb-girdle muscular dystrophy and cardiac arrhythmia. Muscle biopsies of affected patients display impaired membrane trafficking of both POPDC isoforms. Biopsy material of patients carrying mutations in BVES were immunostained with POPDC antibodies. The interaction of POPDC proteins was investigated by co-precipitation, proximity ligation, bioluminescence resonance energy transfer and bimolecular fluorescence complementation. Site-directed mutagenesis was utilised to map the domains involved in protein–protein interaction. Patients carrying a novel homozygous variant, BVES (c.547G > T, p.V183F) displayed only a skeletal muscle pathology and a mild impairment of membrane trafficking of both POPDC isoforms. In contrast, variants such as BVES p.Q153X or POPDC2 p.W188X were associated with a greater impairment of membrane trafficking. Co-transfection analysis in HEK293 cells revealed that POPDC proteins interact with each other through a helix-helix interface located at the C-terminus of the Popeye domain. Site-directed mutagenesis of an array of ultra-conserved hydrophobic residues demonstrated that some of them are required for membrane trafficking of the POPDC1–POPDC2 complex. Mutations in POPDC proteins that cause an impairment in membrane localization affect POPDC complex formation while mutations which leave protein–protein interaction intact likely affect some other essential function of POPDC proteins.

The establishment of macromolecular complexes by scaffolding proteins is key to the local production of cAMP by anchored adenylyl cyclase (AC) and the subsequent cAMP signaling necessary for cardiac functions. We identify a novel AC scaffold, the Popeye domain‐containing (POPDC) protein. The POPDC family of proteins is important for cardiac pacemaking and conduction, due in part to their cAMP‐dependent binding and regulation of TREK‐1 potassium channels. We show that TREK‐1 binds the AC9:POPDC1 complex and copurifies in a POPDC1‐dependent manner with AC9 activity in heart. Although the AC9:POPDC1 interaction is cAMP‐independent, TREK‐1 association with AC9 and POPDC1 is reduced upon stimulation of the β‐adrenergic receptor (βAR). AC9 activity is required for βAR reduction of TREK‐1 complex formation with AC9:POPDC1 and in reversing POPDC1 enhancement of TREK‐1 currents. Finally, deletion of the gene‐encoding AC9 ( Adcy9) gives rise to bradycardia at rest and stress‐induced heart rate variability, a milder phenotype than the loss of Popdc1 but similar to the loss of Kcnk2 (TREK‐1). Thus, POPDC1 represents a novel adaptor for AC9 interactions with TREK‐1 to regulate heart rate control.

Background: The Popeye domain containing (POPDC) genes encode sarcolemma-localised cAMP effector proteins. Mutations in BVES (POPDC1) and POPDC2 have been associated with limb-girdle muscular dystrophy and cardiac arrhythmia. Muscle biopsies of affected patients display impaired membrane trafficking of both POPDC isoforms. Methods: Biopsy material of patients carrying mutations in BVES were immunostained with POPDC antibodies. The interaction of POPDC proteins was investigated by co-precipitation, proximity ligation, bioluminescence resonance energy transfer and bimolecular fluorescence complementation. Site-directed mutagenesis was utilised to map the domains involved in protein interaction. Findings: Patients carrying a novel homozygous variant, BVES (c.547G>T, p.V183F) displayed only a skeletal muscle pathology and a mild impairment of membrane trafficking of both POPDC isoforms. This is in contrast to variants such as BVES p.Q153X or POPDC2 p.W188X, which were associated with a greater impairment of membrane trafficking. Co-transfection analysis in HEK293 cells revealed that POPDC proteins interact with each other through a helix-helix interface located at the C-terminus of the Popeye domain. Site-directed mutagenesis of an array of ultra-conserved hydrophobic residues demonstrated that some of them are required for membrane trafficking of the POPDC1-POPDC2 complex. Interpretation: Mutations in POPDC proteins that cause an impairment in membrane localisation affect POPDC complex formation while mutations which leave the protein interaction intact likely affect some other essential function of POPDC proteins. Funding: This study was funded by an EPSRC/British Heart Foundation co-funded Imperial Institute of Chemical Biology (ICB) Centre for Doctoral Training (CDT) PhD studentship (EP/S023518/1), a project grant of the British Heart Foundation (PG19/13/34247) and the Deutsche Forschungsgemeinschaft (DE1482/9-1). Research in Context: Evidence before this study: Several biallelic missense and nonsense variants in BVES (POPDC1) have been described and are associated with heart and skeletal muscle disease. Skeletal muscle biopsies of homozygous carriers of these variants display a loss of sarcolemmal localisation of POPDC1 and POPDC2. Added value of this study: We demonstrate that POPDC1 and POPDC2 form a heteromeric complex and that complex formation is required for plasma membrane trafficking of POPDC proteins. Transfection of different disease variants in HEK293 cells replicates their defective membrane targeting observed in biopsy material. Structural modelling and site-directed mutagenesis identifies an interface of strongly conserved hydrophobic residues in POPDC proteins, which likely mediate the interaction of POPDC proteins. Implications of all the available evidence: These data provide novel insight into the membrane targeting requirements of POPDC proteins. We recommend testing the membrane targeting properties of any novel variant in POPDC isoforms using a newly developed co-transfection assay in HEK293 cells to characterise its pathogenicity. Our novel insight into the requirement of heterodimerization for proper membrane targeting may also offer novel opportunities to treat patients carrying mutations in POPDC proteins.

Extensive research has uncovered diverse forms of synaptic plasticity and an array of molecular signaling mechanisms that act as positive or negative regulators. Specifically, cyclic 3′,5′-cyclic adenosine monophosphate (cAMP)-dependent signaling pathways are crucially implicated in long-lasting synaptic plasticity. In this study, we examine the role of Popeye domain-containing protein 1 (POPDC1) (or blood vessel epicardial substance (BVES)), a cAMP effector protein, in modulating hippocampal synaptic plasticity. Unlike other cAMP effectors, such as protein kinase A (PKA) and exchange factor directly activated by cAMP, POPDC1 is membrane-bound and the sequence of the cAMP-binding cassette differs from canonical cAMP-binding domains, suggesting that POPDC1 may have an unique role in cAMP-mediated signaling. Our results show that Popdc1 is widely expressed in various brain regions including the hippocampus. Acute hippocampal slices from Popdc1 knockout (KO) mice exhibit PKA-dependent enhancement in CA1 long-term potentiation (LTP) in response to weaker stimulation paradigms, which in slices from wild-type mice induce only transient LTP. Loss of POPDC1, while not affecting basal transmission or input-specificity of LTP, results in altered response during high-frequency stimulation. Popdc1 KO mice also show enhanced forskolin-induced potentiation. Overall, these findings reveal POPDC1 as a novel negative regulator of hippocampal synaptic plasticity and, together with recent evidence for its interaction with phosphodiesterases (PDEs), suggest that POPDC1 is involved in modulating activity-dependent local cAMP–PKA–PDE signaling.