Mutations in LMNA , which encodes the nuclear proteins Lamin A/C, can cause cardiomyopathy and conduction disorders. Here, we employ induced pluripotent stem cells (iPSCs) generated from human cells carrying heterozygous K219T mutation on LMNA to develop a disease model. Cardiomyocytes differentiated from these iPSCs, and which thus carry K219T- LMNA , have altered action potential, reduced peak sodium current and diminished conduction velocity. Moreover, they have significantly downregulated Na v 1.5 channel expression and increased binding of Lamin A/C to the promoter of SCN5A , the channel’s gene. Coherently, binding of the Polycomb Repressive Complex 2 (PRC2) protein SUZ12 and deposition of the repressive histone mark H3K27me3 are increased at SCN5A . CRISPR/Cas9-mediated correction of the mutation re-establishes sodium current density and SCN5A expression. Thus, K219T- LMNA cooperates with PRC2 in downregulating SCN5A , leading to decreased sodium current density and slower conduction velocity. This mechanism may underlie the conduction abnormalities associated with LMNA-cardiomyopathy.

SummaryCardiomyocytes from the apex but not the base of the heart increase their contractility in response to β2-adrenoceptor (β2AR) stimulation, which may underlie the development of Takotsubo cardiomyopathy. However, both cell types produce comparable cytosolic amounts of the second messenger cAMP. We investigated this discrepancy using nanoscale imaging techniques and found that, structurally, basal cardiomyocytes have more organized membranes (higher T-tubular and caveolar densities). Local membrane microdomain responses measured in isolated basal cardiomyocytes or in whole hearts revealed significantly smaller and more short-lived β2AR/cAMP signals. Inhibition of PDE4, caveolar disruption by removing cholesterol or genetic deletion of Cav3 eliminated differences in local cAMP production and equilibrated the contractile response to β2AR. We conclude that basal cells possess tighter control of cAMP because of a higher degree of signaling microdomain organization. This provides varying levels of nanostructural control for cAMP-mediated functional effects that orchestrate macroscopic, regional physiological differences within the heart.

Rationale: Ca 2+ induced Ca 2+ release (CICR) in normal hearts requires close approximation of L-type calcium channels (LTCCs) within the transverse tubules (T-tubules), and Ryanodine receptors (RyR) within the junctional sarcoplasmic reticulum (jSR). CICR is disrupted in cardiac hypertrophy and heart failure, which is associated with loss of T-tubules and disruption of cardiac dyads. In these conditions, LTCCs are redistributed from the T-tubules to disrupt CICR. The molecular mechanism responsible for LTCCs recruitment to and from the T-tubules is not well known. Junctophilin-2 (JPH2) enables close association between T-tubules and the jSR to ensure efficient CICR. JPH2 has a so-called Joining region that is located near domains that interact with T-tubular plasma membrane, where LTCCs are housed. The idea that this Joining region directly interacts with LTCCs and contributes to LTCC recruitment to T-tubules is unknown. Objective: To determine if the Joining region in JPH2 recruits LTCCs to T-tubules through direct molecular interaction in cardiomyocytes to enable efficient CICR. Methods and Results: Modified abundance of JPH2 and redistribution of LTCC were studied in left ventricular hypertrophy in vivo and in cultured adult Feline and rat ventricular myocytes. Protein-protein interaction studies showed that the Joining region in JPH2 interacts with LTCC-α1C subunit and causes LTCCs distribution to the dyads, where they colocalize with RyRs. A JPH2 with induced mutations in the Joining region (mutPG1JPH2) caused T-tubule remodeling and dyad loss, showing that an interaction between LTCC and JPH2 is crucial for T-tubule stabilization. mut PG1JPH2 caused asynchronous Ca 2+ -release with impaired excitation-contraction (EC) coupling after β-adrenergic stimulation. The disturbed Ca 2+ regulation in mut PG1JPH2 overexpressing myocytes caused Calcium/calmodulin-dependent kinase-II activation and altered myocyte bioenergetics. Conclusions: The interaction between LTCC and the Joining region in JPH2 facilitates dyad assembly and maintains normal CIRC in cardiomyocytes.

Aim In cardiomyocytes, transverse tubules (T-tubules) associate with the sarcoplasmic reticulum (SR), forming junctional membrane complexes (JMCs) where L-type calcium channels (LTCCs) are juxtaposed to Ryanodine receptors (RyR). Junctophilin-2 (JPH2) supports the assembly of JMCs by tethering T-tubules to the SR membrane. T-tubule remodelling in cardiac diseases is associated with downregulation of JPH2 expression suggesting that JPH2 plays a crucial role in T-tubule stability. Furthermore, increasing evidence indicate that JPH2 might additionally act as a modulator of calcium signalling by directly regulating RyR and LTCCs. This study aimed at determining whether JPH2 overexpression restores normal T-tubule structure and LTCC function in cultured cardiomyocytes. Methods and results Rat ventricular myocytes kept in culture for 4 days showed extensive T-tubule remodelling with impaired JPH2 localization and relocation of the scaffolding protein Caveolin3 (Cav3) from the T-tubules to the outer membrane. Overexpression of JPH2 restored T-tubule structure and Cav3 relocation. Depletion of membrane cholesterol by chronic treatment with methyl-β-cyclodextrin (MβCD) countered the stabilizing effect of JPH2 overexpression on T-tubules and Cav3. Super-resolution scanning patch-clamp showed that JPH2 overexpression greatly increased the number of functional LTCCs at the plasma membrane. Treatment with MβCD reduced LTCC open probability and activity. Proximity ligation assays showed that MβCD did not affect JPH2 interaction with RyR and the pore-forming LTCC subunit Cav1.2, but strongly impaired JPH2 association with Cav3 and the accessory LTCC subunit Cavβ2. Conclusions JPH2 promotes T-tubule structural stability and recruits functional LTCCs to the membrane, most likely by directly binding to the channel. Cholesterol is involved in the binding of JPH2 to T-tubules as well as in the modulation of LTCC activity. We propose a model where cholesterol and Cav3 support the assembly of lipid rafts which provide an anchor for JPH2 to form JMCs and a platform for signalling complexes to regulate LTCC activity.

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Recurrent cerebral ischemia after PFO closure is more frequent in older patients and could most likely be associated to conditions related to age (atherosclerosis, atrial fibrillation), than to paradoxical embolism. The procedure is as safe as in younger patients.

Objective: Sensory deficits are important risk factors for delirium but have been investigated in single-center studies and single clinical settings. This multicenter study aims to evaluate the association between hearing and visual impairment or bi-sensory impairment (visual and hearing impairment) and delirium. Design: Cross-sectional study nested in the 2017 "Delirium Day" project. Setting and Participants: Patients 65 years and older admitted to acute hospital medical wards, emergency departments, rehabilitation wards, nursing homes, and hospices in Italy. Methods: Delirium was assessed with the 4AT (a short tool for delirium assessment) and sensory deficits with a clinical evaluation. We assessed the association between delirium, hearing and visual impairment in multivariable logistic regression models, adjusting for: Model 1, we included predisposing factors for delirium (ie, dementia, weight loss and autonomy in the activities of daily living); Model 2, we added to Model 1 variables, which could be considered precipitating factors for delirium (ie, psychoactive drugs and urinary catheters). Results: A total of 3038 patients were included; delirium prevalence was 25%. Patients with delirium had a higher prevalence of hearing impairment (30.5% vs 18%; P < .001), visual impairment (24.2% vs 15.7%; P < .01) and bi-sensory impairment (16.2% vs 7.5%) compared with those without delirium. In the multivariable logistic regression analysis, the presence of bi-sensory impairment was associated with delirium in Model 1 [odds ratio (OR) 1.5, confidence interval (CI) 1.2e2.1; P ¼ .00] and in Model 2 (OR 1.4; CI 1.1e1.9; P ¼ .02), whereas the presence of visual and hearing impairment alone was not associated with delirium either in Model 1 (OR 0.8; CI 0.6e1.2, P ¼ .36; OR 1.1; CI 0.8e1.4; P ¼ .42) or in Model 2 (OR 0.8, CI 0.6e1.2, P ¼ .27; OR 1.1, CI 0.8e1.4, P ¼ .63).