Littérature scientifique sur le sujet « PLN-R14Del »

Phospholamban (PLN), a key modulator of Ca2+-homeostasis, inhibits sarcoplasmic reticulum (SR) calcium-ATPase (SERCA2a) and regulates cardiac contractility. The human PLN mutation R14del has been identified in arrhythmogenic cardiomyopathy patients worldwide and is currently extensively investigated. In search of the molecular mechanisms mediating the pathological phenotype, we examined PLN-R14del associations to known PLN-interacting partners. We determined that PLN-R14del interactions to key Ca2+-handling proteins SERCA2a and HS-1-associated protein X-1 (HAX-1) were enhanced, indicating the super-inhibition of SERCA2a’s Ca2+-affinity. Additionally, histidine-rich calcium binding protein (HRC) binding to SERCA2a was increased, suggesting the inhibition of SERCA2a maximal velocity. As phosphorylation relieves the inhibitory effect of PLN on SERCA2a activity, we examined the impact of phosphorylation on the PLN-R14del/SERCA2a interaction. Contrary to PLN-WT, phosphorylation did not affect PLN-R14del binding to SERCA2a, due to a lack of Ser-16 phosphorylation in PLN-R14del. No changes were observed in the subcellular distribution of PLN-R14del or its co-localization to SERCA2a. However, in silico predictions suggest structural perturbations in PLN-R14del that could impact its binding and function. Our findings reveal for the first time that by increased binding to SERCA2a and HAX-1, PLN-R14del acts as an enhanced inhibitor of SERCA2a, causing a cascade of molecular events contributing to impaired Ca2+-homeostasis and arrhythmogenesis. Relieving SERCA2a super-inhibition could offer a promising therapeutic approach for PLN-R14del patients.

Phospholamban (PLN) is a major regulator of cardiac contractility, and human mutations in this gene give rise to inherited cardiomyopathies. The deletion of Arginine 14 is the most-prevalent cardiomyopathy-related mutation, and it has been linked to arrhythmogenesis and early death. Studies in PLN-humanized mutant mice indicated an increased propensity to arrhythmias, but the underlying cellular mechanisms associated with R14del-PLN cardiac dysfunction in the absence of any apparent structural remodeling remain unclear. The present study addressed the specific role of myofilaments in the setting of R14del-PLN and the long-term effects of R14del-PLN in the heart. Maximal force was depressed in skinned cardiomyocytes from both left and right ventricles, but this effect was more pronounced in the right ventricle of R14del-PLN mice. In addition, the Ca2+ sensitivity of myofilaments was increased in both ventricles of mutant mice. However, the depressive effects of R14del-PLN on contractile parameters could be reversed with the positive inotropic drug omecamtiv mecarbil, a myosin activator. At 12 months of age, corresponding to the mean symptomatic age of R14del-PLN patients, contractile parameters and Ca2+ transients were significantly depressed in the right ventricular R14del-PLN cardiomyocytes. Echocardiography did not reveal any alterations in cardiac function or remodeling, although histological and electron microscopy analyses indicated subtle alterations in mutant hearts. These findings suggest that both aberrant myocyte calcium cycling and aberrant contractility remain specific to the right ventricle in the long term. In addition, altered myofilament activity is an early characteristic of R14del-PLN mutant hearts and the positive inotropic drug omecamtiv mecarbil may be beneficial in treating R14del-PLN cardiomyopathy.

Background: Phospholamban (PLN) is a critical regulator of calcium cycling and contractility in the heart. The loss of arginine at position 14 in PLN (R14del) is associated with dilated cardiomyopathy with a high prevalence of ventricular arrhythmias. How the R14 deletion causes dilated cardiomyopathy is poorly understood, and there are no disease-specific therapies. Methods: We used single-cell RNA sequencing to uncover PLN R14del disease mechanisms in human induced pluripotent stem cells (hiPSC-CMs). We used both 2-dimensional and 3-dimensional functional contractility assays to evaluate the impact of modulating disease-relevant pathways in PLN R14del hiPSC-CMs. Results: Modeling of the PLN R14del cardiomyopathy with isogenic pairs of hiPSC-CMs recapitulated the contractile deficit associated with the disease in vitro. Single-cell RNA sequencing revealed the induction of the unfolded protein response (UPR) pathway in PLN R14del compared with isogenic control hiPSC-CMs. The activation of UPR was also evident in the hearts from PLN R14del patients. Silencing of each of the 3 main UPR signaling branches (IRE1, ATF6, or PERK) by siRNA exacerbated the contractile dysfunction of PLN R14del hiPSC-CMs. We explored the therapeutic potential of activating the UPR with a small molecule activator, BiP (binding immunoglobulin protein) inducer X. PLN R14del hiPSC-CMs treated with BiP protein inducer X showed a dose-dependent amelioration of the contractility deficit in both 2-dimensional cultures and 3-dimensional engineered heart tissues without affecting calcium homeostasis. Conclusions: Together, these findings suggest that the UPR exerts a protective effect in the setting of PLN R14del cardiomyopathy and that modulation of the UPR might be exploited therapeutically.

The inherited mutation (R14del) in the calcium regulatory protein phospholamban (PLN) is linked to malignant ventricular arrhythmia with poor prognosis starting at adolescence. However, the underlying early mechanisms that may serve as prognostic factors remain elusive. This study generated humanized mice in which the endogenous gene was replaced with either human wild type or R14del-PLN and addressed the early molecular and cellular pathogenic mechanisms. R14del-PLN mice exhibited stress-induced impairment of atrioventricular conduction, and prolongation of both ventricular activation and repolarization times in association with ventricular tachyarrhythmia, originating from the right ventricle (RV). Most of these distinct electrocardiographic features were remarkably similar to those in R14del-PLN patients. Studies in isolated cardiomyocytes revealed RV-specific calcium defects, including prolonged action potential duration, depressed calcium kinetics and contractile parameters, and elevated diastolic Ca-levels. Ca-sparks were also higher although SR Ca-load was reduced. Accordingly, stress conditions induced after contractions, and inclusion of the CaMKII inhibitor KN93 reversed this proarrhythmic parameter. Compensatory responses included altered expression of key genes associated with Ca-cycling. These data suggest that R14del-PLN cardiomyopathy originates with RV-specific impairment of Ca-cycling and point to the urgent need to improve risk stratification in asymptomatic carriers to prevent fatal arrhythmias and delay cardiomyopathy onset.

Background: Arginine (Arg) 14 deletion (R14del) in the calcium regulatory protein phospholamban (hPLN R14del ) has been identified as a disease-causing mutation in patients with an inherited cardiomyopathy. Mechanisms underlying the early arrhythmogenic phenotype that predisposes carriers of this mutation to sudden death with no apparent structural remodeling remain unclear. Methods: To address this, we performed high spatiotemporal resolution optical mapping of intact hearts from adult knock-in mice harboring the human PLN WT (wildtype [WT], n=12) or the heterozygous human PLN R14del mutation (R14del, n=12) before and after ex vivo challenge with isoproterenol and rapid pacing. Results: Adverse electrophysiological remodeling was evident in the absence of significant structural or hemodynamic changes. R14del hearts exhibited increased arrhythmia susceptibility compared with wildtype. Underlying this susceptibility was preferential right ventricular action potential prolongation that was unresponsive to β-adrenergic stimulation. A steep repolarization gradient at the left ventricular/right ventricular interface provided the substrate for interventricular activation delays and ultimately local conduction block during rapid pacing. This was followed by the initiation of macroreentrant circuits supporting the onset of ventricular tachycardia. Once sustained, these circuits evolved into high-frequency rotors, which in their majority were pinned to the right ventricle. These rotors exhibited unique spatiotemporal dynamics that promoted their increased stability in R14del compared with wildtype hearts. Conclusions: Our findings highlight the crucial role of primary electric remodeling caused by the hPLN R14del mutation. These inherently arrhythmogenic features form the substrate for adrenergic-mediated VT at early stages of PLN R14del induced cardiomyopathy.

Inherited cardiomyopathy caused by the p.(Arg14del) pathogenic variant of the phospholamban (PLN) gene is characterized by intracardiomyocyte PLN aggregation and can lead to severe dilated cardiomyopathy. We recently reported that pre-emptive depletion of PLN attenuated heart failure (HF) in several cardiomyopathy models. Here, we investigated if administration of a Pln-targeting antisense oligonucleotide (ASO) could halt or reverse disease progression in mice with advanced PLN-R14del cardiomyopathy. To this aim, homozygous PLN-R14del (PLN-R14 Δ/Δ) mice received PLN-ASO injections starting at 5 or 6 weeks of age, in the presence of moderate or severe HF, respectively. Mice were monitored for another 4 months with echocardiographic analyses at several timepoints, after which cardiac tissues were examined for pathological remodeling. We found that vehicle-treated PLN-R14 Δ/Δ mice continued to develop severe HF, and reached a humane endpoint at 8.1 ± 0.5 weeks of age. Both early and late PLN-ASO administration halted further cardiac remodeling and dysfunction shortly after treatment start, resulting in a life span extension to at least 22 weeks of age. Earlier treatment initiation halted disease development sooner, resulting in better heart function and less remodeling at the study endpoint. PLN-ASO treatment almost completely eliminated PLN aggregates, and normalized levels of autophagic proteins. In conclusion, these findings indicate that PLN-ASO therapy may have beneficial outcomes in PLN-R14del cardiomyopathy when administered after disease onset. Although existing tissue damage was not reversed, further cardiomyopathy progression was stopped, and PLN aggregates were resolved.

The deletion of the arginine 14 codon (R14del) in the phospholamban (PLN) gene is a rare cause of arrhythmogenic cardiomyopathy (ACM) and is associated with prevalent ventricular arrhythmias, heart failure, and sudden cardiac death. The pathophysiological mechanism which culminates in the ACM phenotype is multifactorial and mainly based on the alteration of the endoplasmic reticulum proteostasis, mitochondrial dysfunction and compromised Ca2+ cytosolic homeostasis. The symptoms of this condition are usually non-specific and consist of arrhythmia-related or heart failure-related manifestation; however, some peculiar diagnostic clues were detected, such as the T-wave inversion in the lateral leads, low QRS complexes voltages, mid-wall or epicardial fibrosis of the inferolateral wall of the left ventricle, and their presence should raise the suspicion of this condition. The risk stratification for sudden cardiac death is mandatory and several predictors were identified in recent years. However, the management of affected patients is often challenging due to the absence of specific prediction tools and therapies. This review aims to provide the current state of the art of PLN R14del cardiomyopathy, focusing on its pathophysiology, clinical manifestation, risk stratification for sudden cardiac death, and management.

Introduzione. L’interazione tra ripolarizzazione ventricolare e il maneggiamento del calcio intracellulare è cruciale nel mantenimento fisiologico dell’accoppiamento eccitazione-contrazione (E-C) nei cardiomiociti. Sia il calcio intracellulare che il riempimento del reticolo sarcoplasmatico (RS) giocano un ruolo importante in questo meccanismo, oltre ad essere influenzati e controllati da molti altri fattori. Tra questi, il prolungamento del potenziale d’azione non fisiologico influenza il bilancio tra calcio che entra/esce dal reticolo, rappresentando quindi un importante fattore di stress per l’omeostasi del calcio intracellulare, richiedendo robusti meccanismi compensatori. Questo può diventare un problema in condizioni patologiche, come nello scompenso cardiaco, in cui il maneggiamento del calcio intracellulare è compromesso. Infatti, condizioni patologiche in cui tale maneggiamento è compromesso può rappresentare un trigger d’induzione di meccanismi aritmogeni. D’altra parte, il prolungamento della ripolarizzazione e le aritmie sono spesso associate fra loro, nonostante la presenza concomitante di altri fattori, come peptidi o polimorfismi di proteine coinvolte nel meccanismo di E-C, possono essere necessari per portare ad aritmogenesi. Scopo. In questa tesi, gli scopi sono: 1) capire il ruolo e i meccanismi dell’angiotensina II e dei polimorfismi di NOS1AP nel generare instabilità reticolare in presenza di ripolarizzazione prolungata; 2) indagare sui meccanismi di accoppiamento E-C nei cardiomiociti umani derivati da staminali pluripotenti indotte che portano la mutazione del fosfolambano (R14Del) tramite l’uso di un farmaco lusitropo e inotropo sviluppato nel nostro laboratorio. Risultati e discussione. I risultati ottenuti in questa tesi hanno confermato che il prolungamento della durata del potenziale d’azione da solo non è sufficiente per indurre aritmie, pertanto, per indurre aritmogenesi, è necessaria la presenza di fattori concomitanti, come l’angiotensina II o polimorfismi di NOS1AP. Allo stesso modo, altri meccanismi, non strettamente correlati al RS, sembrano essere coinvolti nella generazione del fenotipo dei pazienti con scompenso cardiaco affetti dalla mutazione del fosfolambano R14Del. Lo studio dei meccanismi coinvolti nelle patologie genetiche correlate al RS ha valore fondamentale e traslazionale, che potrebbe essere utile per aiutare i pazienti affetti da malattie cardiache.
Background. The interplay between ventricular repolarization and intracellular Ca2+ handling is crucial to maintain the physiological excitation-contraction coupling (ECC) in cardiac myocytes. The intracellular Ca2+ and the sarcoplasmic reticulum (SR) Ca2+ load play a crucial role in this mechanism and they are influenced, and controlled, by several factors. Among them, the non-physiological action potential duration (APD) prolongation affects the sarcolemmal Ca2+ influx/efflux balance thus it represents a stress-condition for intracellular Ca2+ homeostasis, requiring robust compensatory mechanisms. This may be critical in pathological conditions, such as heart failure (HF), where intracellular Ca2+ handling is impaired. Indeed, pathophysiological conditions in which the SR Ca2+ handling is altered could represent a trigger to induce arrhythmogenic afterdepolarizations. On the other hand, prolonged repolarization and arrhythmias are often associated, albeit the concomitance of multiple factors, such as peptides or polymorphisms in proteins involved in the ECC, could be necessary to produce arrhythmogenesis. Aims. In this thesis, the purposes are: 1) to address the role and mechanisms of angiotensin II and NOS1AP polymorphisms in generating SR instability in the presence of prolonged repolarization; 2) to investigate the ECC mechanisms in human-induced pluripotent stem-cell derived cardiomyocytes (hiPSC-CMs) carrying the phospholamban (PLN) mutation R14Del, by the use of a luso-inotropic drug developed in our lab. Results and discussion. The results obtained have confirmed that APD prolongation per se could be insufficient to induce arrhythmias thus the presence of concomitant factors, such as angiotensin II or NOS1AP polymorphisms, are necessary to produce arrhytmogenesis. Similarly, other mechanisms, not strictly correlated to the SR, seem to be involved in the generation of the phenotype of HF patients affected by the PLN-R14Del mutation. The study of mechanisms involved in SR-correlated genetic pathologies, have both a fundamental and translational value, which could be useful to help patients affected by cardiac diseases.