Journal articles: 'PLN-R14Del'

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Vafiadaki, Elizabeth, Kobra Haghighi, Demetrios A. Arvanitis, Evangelia G. Kranias, and Despina Sanoudou. "Aberrant PLN-R14del Protein Interactions Intensify SERCA2a Inhibition, Driving Impaired Ca2+ Handling and Arrhythmogenesis." International Journal of Molecular Sciences 23, no. 13 (June 22, 2022): 6947. http://dx.doi.org/10.3390/ijms23136947.

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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.

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Kumar, Mohit, Kobra Haghighi, Sheryl Koch, Jack Rubinstein, Francesca Stillitano, Roger J. Hajjar, Evangelia G. Kranias, and Sakthivel Sadayappan. "Myofilament Alterations Associated with Human R14del-Phospholamban Cardiomyopathy." International Journal of Molecular Sciences 24, no. 3 (January 31, 2023): 2675. http://dx.doi.org/10.3390/ijms24032675.

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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.

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Feyen, Dries A. M., Isaac Perea-Gil, Renee G. C. Maas, Magdalena Harakalova, Alexandra A. Gavidia, Jennifer Arthur Ataam, Ting-Hsuan Wu, et al. "Unfolded Protein Response as a Compensatory Mechanism and Potential Therapeutic Target in PLN R14del Cardiomyopathy." Circulation 144, no. 5 (August 3, 2021): 382–92. http://dx.doi.org/10.1161/circulationaha.120.049844.

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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.

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Haghighi, Kobra, George Gardner, Elizabeth Vafiadaki, Mohit Kumar, Lisa C. Green, Jianyong Ma, Jeffrey S. Crocker, et al. "Impaired Right Ventricular Calcium Cycling Is an Early Risk Factor in R14del-Phospholamban Arrhythmias." Journal of Personalized Medicine 11, no. 6 (June 3, 2021): 502. http://dx.doi.org/10.3390/jpm11060502.

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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.

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Raad, Nour, Philip Bittihn, Marine Cacheux, Dongtak Jeong, Zeki Ilkan, Delaine Ceholski, Erik Kohlbrenner, et al. "Arrhythmia Mechanism and Dynamics in a Humanized Mouse Model of Inherited Cardiomyopathy Caused by Phospholamban R14del Mutation." Circulation 144, no. 6 (August 10, 2021): 441–54. http://dx.doi.org/10.1161/circulationaha.119.043502.

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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.

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Eijgenraam, Tim R., Nienke M. Stege, Vivian Oliveira Nunes Teixeira, Remco de Brouwer, Elisabeth M. Schouten, Niels Grote Beverborg, Liu Sun, et al. "Antisense Therapy Attenuates Phospholamban p.(Arg14del) Cardiomyopathy in Mice and Reverses Protein Aggregation." International Journal of Molecular Sciences 23, no. 5 (February 22, 2022): 2427. http://dx.doi.org/10.3390/ijms23052427.

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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.

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Badone, Beatrice, Carlotta Ronchi, Francesco Lodola, Claudia Maniezzi, Daniele Martone, Anika E. Knaust, Thomas Eschenhagen, Arne Hansen, and Antonio Zaza. "Characterization of the PLN-R14Del mutation in hiPSC-derived cardiomyocytes." Vascular Pharmacology 146 (October 2022): 107054. http://dx.doi.org/10.1016/j.vph.2022.107054.

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Badone, Beatrice, Carlotta Ronchi, Francesco Lodola, Claudia Maniezzi, Daniele Martone, Anika E. Knaust, Arne Hansen, Thomas Eschenaghen, and Antonio Zaza. "Characterization of the PLN-R14Del mutation in hiPSC-derived cardiomyocytes." Biophysical Journal 121, no. 3 (February 2022): 91a. http://dx.doi.org/10.1016/j.bpj.2021.11.2271.

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Monda, Emanuele, Ettore Blasi, Antonio De Pasquale, Alessandro Di Vilio, Federica Amodio, Martina Caiazza, Gaetano Diana, et al. "Clinical and Molecular Characteristics of Patients with PLN R14del Cardiomyopathy: State-of-the-Art Review." Cardiogenetics 12, no. 1 (March 2, 2022): 112–21. http://dx.doi.org/10.3390/cardiogenetics12010012.

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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.

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Mittal, Nishka, Jaydev Dave, Magdalena Harakalova, J. Peter van Tintelen, Folkert W. Asselbergs, Pieter A. Doevendans, Kevin D. Costa, Irene C. Turnbull, and Francesca Stillitano. "Generation of human induced pluripotent stem cell (iPSC) lines derived from five patients carrying the pathogenic phospholamban-R14del (PLN-R14del) variant and three non-carrier family members." Stem Cell Research 60 (April 2022): 102737. http://dx.doi.org/10.1016/j.scr.2022.102737.

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Maniezzi, Claudia, Daniele Martone, Marem Eskandr, Beatrice Ferè, Chiara Florindi, Herman Silljé, Rudolf de Boer, Francesco Lodola, and Antonio Zaza. "Unraveling the pathophysiological role of the PLN-R14del mutation in a novel heterozygous mouse model of dilated cardiomyopathy." Vascular Pharmacology 146 (October 2022): 107056. http://dx.doi.org/10.1016/j.vph.2022.107056.

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Lodola, Francesco, Claudia Maniezzi, Marem Eskandr, Chiara Florindi, Rudolf A. de Boer, Herman Silljé, and Antonio Zaza. "Exploring the pathophysiological role of the PLN-R14del mutation in a novel heterozygous mouse model of dilated cardiomyopathy." Biophysical Journal 122, no. 3 (February 2023): 382a. http://dx.doi.org/10.1016/j.bpj.2022.11.2096.

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Ceholski, Delaine K., Irene C. Turnbull, Simon Koplev, Chi-Wing Kong, Francesca Stillitano, Mathieu Nonnenmacher, Kobra Haghighi, et al. "Abstract 185: Genome Editing of Isogenic Human Induced Pluripotent Stem Cells Allows for Functional and Transcriptomic Insights Into Hereditary Dilated Cardiomyopathy Caused by Phospholamban Mutations." Circulation Research 121, suppl_1 (July 21, 2017). http://dx.doi.org/10.1161/res.121.suppl_1.185.

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Dilated cardiomyopathy (DCM) can be caused by genetic mutations in numerous cardiac proteins, including phospholamban (PLN). PLN mutations are quite rare and obtaining patient samples for mechanistic insights can be challenging. We used genome editing with CRISPR/Cas9 to successfully insert R14del and R9C PLN mutations into a human induced pluripotent stem cell (hiPSC) line from an individual with no cardiovascular disease. HiPSC-cardiomyocytes (hiPSC-CMs) with the inserted R14del PLN mutation recapitulate the phenotype observed in patient-derived R14del hiPSC-CMs, characterized by abnormal intracellular calcium cycling and arrhythmogenicity. Insertion of R9C PLN results in hiPSC-CMs displaying an abnormal response to β-agonists, defective calcium handling, and a hypertrophic phenotype. In human engineered cardiac tissues (hECTs) created from hiPSC-CMs in a 3D matrix, R14del results in a progressive worsening of developed force and R9C PLN demonstrates an abnormal lusitropic response following β-adrenergic stimulation. Further, transcriptional profiling using RNAseq suggests a role for lipid metabolism in R14del. DNA methylation studies showed that differentially expressed genes were enriched for lipoprotein metabolism and chylomicron-mediated lipid transport pathways in R14del PLN. This was also confirmed with the observation of lipid deposition in R14del hECTs and human myocardial tissues from explanted hearts of affected patients. Furthermore, small RNAseq identified 2 miRNAs that were differentially regulated in R14del hiPSC-CMs (miR-449c-5p and miR-483-3p). For R9C PLN, RNAseq suggests that the mutation results in profibrotic signaling, activation of autophagy, and an altered metabolic state. Our findings demonstrate that gene editing of hiPSCs can be used to successfully create models and delineate molecular mechanisms of human PLN mutations associated with DCM.

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Dave, Jaydev, Nour Raad, Nishka Mittal, Lu Zhang, Anthony Fargnoli, Jae Gyun Oh, Maria Elisabetta Savoia, et al. "Gene editing reverses arrhythmia susceptibility in humanized PLN-R14del mice: modelling a European cardiomyopathy with global impact." Cardiovascular Research, February 22, 2022. http://dx.doi.org/10.1093/cvr/cvac021.

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Abstract Aims A mutation in the phospholamban (PLN) gene, leading to deletion of Arg14 (R14del), has been associated with malignant arrhythmias and ventricular dilation. Identifying pre-symptomatic carriers with vulnerable myocardium is crucial because arrhythmia can result in sudden cardiac death, especially in young adults with PLN-R14del mutation. This study aimed at assessing the efficiency and efficacy of in vivo genome editing, using CRISPR/Cas9 and a cardiotropic adeno-associated virus-9 (AAV9), in improving cardiac function in young adult mice expressing the human PLN-R14del. Methods and results Humanized mice were generated expressing human wild-type (hPLN-WT) or mutant (hPLN-R14del) PLN in the heterozygous state, mimicking human carriers. Cardiac magnetic resonance imaging at 12 weeks of age showed bi-ventricular dilation and increased stroke volume in mutant vs. WT mice, with no deficit in ejection fraction or cardiac output. Challenge of ex vivo hearts with isoproterenol and rapid pacing unmasked higher propensity for sustained ventricular tachycardia (VT) in hPLN-R14del relative to hPLN-WT. Specifically, the VT threshold was significantly reduced (20.3 ± 1.2 Hz in hPLN-R14del vs. 25.7 ± 1.3 Hz in WT, P < 0.01) reflecting higher arrhythmia burden. To inactivate the R14del allele, mice were tail-vein-injected with AAV9.CRISPR/Cas9/gRNA or AAV9 empty capsid (controls). CRISPR-Cas9 efficiency was evaluated by droplet digital polymerase chain reaction and NGS-based amplicon sequencing. In vivo gene editing significantly reduced end-diastolic and stroke volumes in hPLN-R14del CRISPR-treated mice compared to controls. Susceptibility to VT was also reduced, as the VT threshold was significantly increased relative to controls (30.9 ± 2.3 Hz vs. 21.3 ± 1.5 Hz; P < 0.01). Conclusions This study is the first to show that disruption of hPLN-R14del allele by AAV9-CRISPR/Cas9 improves cardiac function and reduces VT susceptibility in humanized PLN-R14del mice, offering preclinical evidence for translatable approaches to therapeutically suppress the arrhythmogenic phenotype in human patients with PLN-R14del disease.

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Eijgenraam, Tim R., Bastiaan J. Boukens, Cornelis J. Boogerd, E. Marloes Schouten, Cees W. A. van de Kolk, Nienke M. Stege, Wouter P. te Rijdt, et al. "The phospholamban p.(Arg14del) pathogenic variant leads to cardiomyopathy with heart failure and is unresponsive to standard heart failure therapy." Scientific Reports 10, no. 1 (June 17, 2020). http://dx.doi.org/10.1038/s41598-020-66656-9.

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Abstract Phospholamban (PLN) plays a role in cardiomyocyte calcium handling as primary inhibitor of sarco/endoplasmic reticulum Ca2+-ATPase (SERCA). The p.(Arg14del) pathogenic variant in the PLN gene results in a high risk of developing dilated or arrhythmogenic cardiomyopathy with heart failure. There is no established treatment other than standard heart failure therapy or heart transplantation. In this study, we generated a novel mouse model with the PLN-R14del pathogenic variant, performed detailed phenotyping, and tested the efficacy of established heart failure therapies eplerenone or metoprolol. Heterozygous PLN-R14del mice demonstrated increased susceptibility to ex vivo induced arrhythmias, and cardiomyopathy at 18 months of age, which was not accelerated by isoproterenol infusion. Homozygous PLN-R14del mice exhibited an accelerated phenotype including cardiac dilatation, contractile dysfunction, decreased ECG potentials, high susceptibility to ex vivo induced arrhythmias, myocardial fibrosis, PLN protein aggregation, and early mortality. Neither eplerenone nor metoprolol administration improved cardiac function or survival. In conclusion, our novel PLN-R14del mouse model exhibits most features of human disease. Administration of standard heart failure therapy did not rescue the phenotype, underscoring the need for better understanding of the pathophysiology of PLN-R14del-associated cardiomyopathy. This model provides a great opportunity to study the pathophysiology, and to screen for potential therapeutic treatments.

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Eijgenraam, Tim R., Cornelis J. Boogerd, Nienke M. Stege, Vivian Oliveira Nunes Teixeira, Martin M. Dokter, Lukas E. Schmidt, Xiaoke Yin, et al. "Protein Aggregation Is an Early Manifestation of Phospholamban p.(Arg14del)–Related Cardiomyopathy: Development of PLN-R14del–Related Cardiomyopathy." Circulation: Heart Failure 14, no. 11 (November 2021). http://dx.doi.org/10.1161/circheartfailure.121.008532.

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Background: The p.(Arg14del) pathogenic variant (R14del) of the PLN (phospholamban) gene is a prevalent cause of cardiomyopathy with heart failure. The exact underlying pathophysiology is unknown, and a suitable therapy is unavailable. We aim to identify molecular perturbations underlying this cardiomyopathy in a clinically relevant PLN-R14del mouse model. Methods: We investigated the progression of cardiomyopathy in PLN-R14 Δ/Δ mice using echocardiography, ECG, and histological tissue analysis. RNA sequencing and mass spectrometry were performed on cardiac tissues at 3 (before the onset of disease), 5 (mild cardiomyopathy), and 8 (end stage) weeks of age. Data were compared with cardiac expression levels of mice that underwent myocardial ischemia-reperfusion or myocardial infarction surgery, in an effort to identify alterations that are specific to PLN-R14del–related cardiomyopathy. Results: At 3 weeks of age, PLN-R14 Δ/Δ mice had normal cardiac function, but from the age of 4 weeks, we observed increased myocardial fibrosis and impaired global longitudinal strain. From 5 weeks onward, ventricular dilatation, decreased contractility, and diminished ECG voltages were observed. PLN protein aggregation was present before onset of functional deficits. Transcriptomics and proteomics revealed differential regulation of processes involved in remodeling, inflammation, and metabolic dysfunction, in part, similar to ischemic heart disease. Altered protein homeostasis pathways were identified exclusively in PLN-R14 Δ/Δ mice, even before disease onset, in concert with aggregate formation. Conclusions: We mapped the development of PLN-R14del–related cardiomyopathy and identified alterations in proteostasis and PLN protein aggregation among the first manifestations of this disease, which could possibly be a novel target for therapy.

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Jiang, Xincheng, Yuanwei Xu, Jiayu Sun, Lili Wang, Xinli Guo, and Yucheng Chen. "The phenotypic characteristic observed by cardiac magnetic resonance in a PLN-R14del family." Scientific Reports 10, no. 1 (October 5, 2020). http://dx.doi.org/10.1038/s41598-020-73359-8.

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Abstract Phospholamban (PLN) is an important regulator for sarcoendoplasmic reticulum (SR) calcium transport ATPase (SERCA), which uptakes Ca2+ to SR during the diastolic phase of cardiomyocytes to maintain intracellular calcium homeostasis. Mutations on PLN result in intracellular calcium disorder, myocardial contraction defect, and eventually heart failure and/or malignant ventricular arrhythmia. Since 2003, several kinds of PLN mutations have been identified in familial dilated cardiomyopathy (DCM) patients, illustrating a few clinical characteristics that differs from classical DCM patients. Herein, we report a large PLN-R14del family with typical clinical characteristics reported including relatively late-onset clinical symptoms, low-voltage in ECG, as well as frequent ventricular arrythmias. Moreover, members underwent cardiac magnetic resonance (CMR) examination showed a strikingly similar pattern of late gadolinium enhancement (LGE)—Sub-epicardial involvement in the left ventricular (LV) lateral wall with or without linear mid-wall enhancement in the interventricular septum. The former one can also present in younger PLN-R14del carriers despite completely normal LV structure and function. Meanwhile, T1 mapping also found significantly increased extracellular volume (ECV) in PLN-R14del carriers. These findings highlight the special role of CMR to phenotyping PLN-induced cardiomyopathy patients and distinguish them from other types of cardiomyopathy.

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Badone, Beatrice, Carlotta Ronchi, Francesco Lodola, Claudia Maniezzi, Marem Eskandr, Chiara Florindi, Anika E. Knaust, Thomas Eschenhagen, Arne Hansen, and Antonio Zaza. "SERCA2a gain of function in patient-derived R14Del hiPSC-CMs." Journal of General Physiology 154, no. 9 (November 12, 2021). http://dx.doi.org/10.1085/jgp.2021ecc3.

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Phospholamban (PLN) is the natural inhibitor of the sarco/endoplasmic reticulum Ca2+ ATPase (SERCA2a). Heterozygous PLN-R14del mutation is associated with an arrhythmogenic dilated cardiomyopathy (DCM), whose pathogenesis has been attributed to SERCA2a “superinhibition.” The aim of the project is to test in human induced pluripotent stem cell–derived cardiomyocytes (hiPSC-CM) harvested from a PLN-R14del carrier whether (1) Ca2+ dynamics and protein localization were compatible with SERCA2a superinhibition and (2) functional abnormalities could be reverted by pharmacological SERCA2a activation with PST3093. Ca2+ transients (CaT) were recorded at 36°C in hiPSC-CMs clusters during field stimulation. SERCA2a and PLN were immunolabeled in single hiPSC-CMs. Mutant (MUT) preparations were compared with isogenic WT ones obtained by mutation reversal. WT and MUT differed for the following properties: (1) CaT time to peak (tpeak) and half-time of CaT decay were shorter in MUT, (2) several CaT profiles were identified in WT, whereas “hyperdynamic” ones largely prevailed in MUT, (3) whereas tpeak rate-dependently declined in WT, it was shorter and rate independent in MUT, and (4) diastolic Ca2+ rate-dependently accumulated in WT, but not in MUT. When applied to WT, PST3093 changed all of the above properties to resemble those of MUT; when applied to MUT, PST3093 had no effect. Preferential perinuclear SERCA2a-PLN localization was lost in MUT hiPSC-CMs. In conclusion, functional data converge to argue for PLN-R14del incompetence in inhibiting SERCA2a in the tested case, thus weakening the rationale for therapeutic SERCA2a activation. Mechanisms alternative to SERCA2a superinhibition should be considered in the pathogenesis of DCM, including dysregulation of Ca2+-dependent transcription.

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Taha, K., W. P. Te Rijdt, H. A. C. M. De Bruin-Bon, M. J. M. Cramer, F. W. Asselbergs, B. J. Bouma, M. P. Van Den Berg, and A. J. Teske. "1041 Early detection of biventricular mechanical dysfunction in PLN R14del mutation carriers." European Heart Journal - Cardiovascular Imaging 21, Supplement_1 (January 1, 2020). http://dx.doi.org/10.1093/ehjci/jez319.633.

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Abstract Funding Acknowledgements PLN Genetic Heart Disease Foundation Background Carriers of the phospholamban (PLN) R14del founder mutation may develop an arrhythmogenic and/or dilated cardiomyopathy. Overt disease is preceded by a pre-symptomatic phase of variable length in which structural abnormalities seem to be absent. Purpose We aimed to explore echocardiographic characteristics of PLN R14del mutation carriers, particularly in early disease stages. Methods We included 120 PLN R14del mutation carriers and classified them to the pre-symptomatic stage (no symptoms and no structural disease, n = 60), the arrhythmic stage (arrhythmic symptoms and left ventricular ejection fraction (LVEF) ≥50%, n = 30) or the structural stage (LVEF <50%, n = 30). We included 60 healthy control subjects who were age- and gender matched with pre-symptomatic mutation carriers. All subjects underwent comprehensive echocardiographic analysis, including deformation imaging. Results Values are provided in the abstract table. Patients in the structural stage had significantly impaired left/right ventricular (LV/RV) function and increased LV/RV size when compared to the other mutation carriers (p < 0.001). In the pre-symptomatic and arrhythmic stage, LV function and volumes did not differ significantly from controls by conventional measurements. However, LV global longitudinal strain (GLS) and LV mechanical dispersion (MD) were already significantly impaired in the pre-symptomatic and arrhythmic stage when compared to controls (p < 0.001). RV function by conventional measurements was lower in arrhythmic subjects than in controls (p = 0.016). A strong linear correlation was found between LV GLS and RV GLS (r = 0.8, p < 0.001). Conclusion Echocardiographic deformation imaging reveals biventricular mechanical alterations in PLN R14del mutation carriers before arrhythmic symptoms and overt structural disease. Longitudinal studies are needed to determine the incremental prognostic value of these findings. Control subjects (n = 60) Pre-symptomatic (n = 60) Arrhythmic (n = 30) Strucutral (n = 30) LVEF(%) 60.3 ± 4.2 58.6 ± 4.2 57.1 ± 5.3 38.8 ± 10.6* LVEDV (ml/m2) 54.6 ± 10.0 53.7 ± 9.5 56.1 ± 11.6 70.1 ± 25.8* LV GLS (%) 21.5 ± 1.8 19.5 ± 1.5* 19.1 ± 1.7* 12.7 ± 3.8* LV MD (msec) 24.4 ± 5.9 33.1 ± 9.5* 48.2 ± 13.3* 60.8 ± 17.1* RV FAC (%) 46.4 ± 4.9 44.7 ± 5.0 42.6 ± 7.4* 33.2 ± 8.0* RV GLS (%) 26.5 ± 3.7 24.2 ± 2.9* 22.5 ± 4.2* 15.1 ± 5.7* FAC = Fractional area change; GLS = Global longitudinal strain; LV/RV = Left/right ventricular; LVEDV = Left ventricular end-diastolic volume; LVEF = Left ventricular ejection fraction; MD = Mechanical dispersion. *p < 0.05 when compared to control subjects. Abstract 1041 Figure. Clinical stages in PLN mutation carriers

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Grote Beverborg, Niels, Daniela Später, Ralph Knöll, Alejandro Hidalgo, Steve T. Yeh, Zaher Elbeck, Herman H. W. Silljé, et al. "Phospholamban antisense oligonucleotides improve cardiac function in murine cardiomyopathy." Nature Communications 12, no. 1 (August 30, 2021). http://dx.doi.org/10.1038/s41467-021-25439-0.

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AbstractHeart failure (HF) is a major cause of morbidity and mortality worldwide, highlighting an urgent need for novel treatment options, despite recent improvements. Aberrant Ca2+ handling is a key feature of HF pathophysiology. Restoring the Ca2+ regulating machinery is an attractive therapeutic strategy supported by genetic and pharmacological proof of concept studies. Here, we study antisense oligonucleotides (ASOs) as a therapeutic modality, interfering with the PLN/SERCA2a interaction by targeting Pln mRNA for downregulation in the heart of murine HF models. Mice harboring the PLN R14del pathogenic variant recapitulate the human dilated cardiomyopathy (DCM) phenotype; subcutaneous administration of PLN-ASO prevents PLN protein aggregation, cardiac dysfunction, and leads to a 3-fold increase in survival rate. In another genetic DCM mouse model, unrelated to PLN (Cspr3/Mlp−/−), PLN-ASO also reverses the HF phenotype. Finally, in rats with myocardial infarction, PLN-ASO treatment prevents progression of left ventricular dilatation and improves left ventricular contractility. Thus, our data establish that antisense inhibition of PLN is an effective strategy in preclinical models of genetic cardiomyopathy as well as ischemia driven HF.

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Deiman, Frederik E., Nils Bomer, Peter van der Meer, and Niels Grote Beverborg. "Review: Precision Medicine Approaches for Genetic Cardiomyopathy: Targeting Phospholamban R14del." Current Heart Failure Reports, June 14, 2022. http://dx.doi.org/10.1007/s11897-022-00558-x.

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Abstract Purpose of Review Heart failure is a syndrome with poor prognosis and no curative options for the majority of patients. The standard one-size-fits-all-treatment approach, targeting neurohormonal dysregulations, helps to modulate symptoms of heart failure, but fails to address the cause of the problem. Precision medicine aims to go beyond symptom modulation and targets pathophysiological mechanisms that underlie disease. In this review, an overview of how precision medicine can be approached as a treatment strategy for genetic heart disease will be discussed. PLN R14del, a genetic mutation known to cause cardiomyopathy, will be used as an example to describe the potential and pitfalls of precision medicine. Recent Findings PLN R14del is characterized by several disease hallmarks including calcium dysregulation, metabolic dysfunction, and protein aggregation. The identification of disease-related biological pathways and the effective targeting using several modalities, including gene silencing and signal transduction modulation, may eventually provide novel treatments for genetic heart disease. Summary We propose a workflow on how to approach precision medicine in heart disease. This workflow focuses on deep phenotyping of patient derived material, including in vitro disease modeling. This will allow identification of therapeutic targets and disease modifiers, to be used for the identification of novel biomarkers and the development of precision medicine approaches for genetic cardiomyopathies.

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Maas, R., S. Lee, M. Harakalova, W. R. Goodyer, P. F. M. Doevendans, J. Van Der Velden, F. W. Asselbergs, J. P. G. Sluijter, S. M. Wu, and J. B. Buikema. "Massive expansion of human induced pluripotent stem cells resulting in efficient biobanking and functional 3D tissue analysis of genetic cardiomyopathies." European Heart Journal 42, Supplement_1 (October 1, 2021). http://dx.doi.org/10.1093/eurheartj/ehab724.3191.

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Abstract Introduction Over the past decade, various protocols were established to ensure efficient differentiation of hiPSC into cardiomyocytes (CMs). A major limitation, however, remained the batch-to-batch variability of hiPSC-CM efficiency and cell number. Here, we suggest an approach in which concomitant GSK-3β inhibition and removal of cell-cell contact inhibition, resulted in a massive proliferative response of hiPSC-CMs1–3. This efficient method allows expansion and passaging of functional hiPSC-CMs, that routinely can be cryopreserved and subsequently used as a stable cell source for the downstream applications, such 3D in vitro models for the disease modelling of dilated cardiomyopathy (DCM). We focussed on the deletion of arginine 14 in the PLN gene (R14del), which is associated with severe heart failure in DCM patients, associated with arrhythmias, cardiac fibrosis and premature death. Methods Subsequent expansion of hiPSC-CM cultures is generally modest (<10 fold). Here, we describe a cost-effective strategy for massive expansion (up to 250-fold) of high-purity hiPSC-CMs relying on two aspects; 1) inhibition of cell-cell contact via low-density seeding and serial passaging in culture flask-format, 2)small molecular glycogen synthase kinase-3β inhibition with CHIR99021 (CHIR). Patient-specific hiPSC-CMs harbouring a PLNR14del mutation were generated and used for EHT formation and functional follow-up. Results We observed that proliferating hiPSC-CMs, especially within the first 2 passages, can routinely be cryopreserved and subsequently further expanded or utilized in downstream applications. Moreover, using this strategy, it is possible to produce ultimately >1 billion CMs within 3–5 weeks starting with one differentiation batch of day 11 hiPSC-CMs, without the need for cell sorting or selection. Expanded hiPSC-CMs retain their capacity to mature and allows fibrin-based engineered heart tissues (EHTs) formation. Previously expanded CMs from PLNR14del patient-specific hiPSC were used to generate EHT and displayed a reduced force phenotype (0.137±0.012 mN) vs healthy control (0.229±0.030 mN) and isogenic control (0.224±0.008 mN) in previously expanded CMs. Conclusion We provpresent a novel strategy for the massive expansion of functional hiPSC-CMs with concomitant GSK-3β inhibition and low cell density culture that ultimately generates up to a 250-fold increase in hiPSC-CM numbers. Expansion healthy control hiPSC-CMs does not limit the subsequent maturation process, and moreover cells remain fully functional such as required for downstream tissue engineering approaches. Therefore, CM expansion forms a well-controlled platform for upscaling hiPSC-CM production for functional 3-dimensionale PLN cardiac disease models, large drug screenings and multiple translational/regenerative applications. Funding Acknowledgement Type of funding sources: Foundation. Main funding source(s): PLN Foundation

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Journal articles on the topic 'PLN-R14Del'

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