Dataset Information

Protein aggregation is an early manifestation of phospholamban p.(Arg14del)-related cardiomyopathy

ABSTRACT: Background: The p.(Arg14del) pathogenic variant (R14del) of the phospholamban (PLN) 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 progression of cardiomyopathy in PLN-R14∆/∆ mice using echocardiography, electrocardiography and histological tissue analysis. RNA sequencing and mass spectrometry were performed on cardiac tissues at 3 weeks of age (before onset of disease), 5 weeks, when mild cardiomyopathy has developed, and 8 weeks (end-stage). 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∆/∆ 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 onwards, ventricular dilatation, decreased contractility and diminished ECG voltages were observed. Strikingly, PLN protein aggregation was present prior to onset of functional deficits. Transcriptomics and proteomics revealed differential regulation of processes involved in remodelling, inflammation and metabolic dysfunction, in part similar to ischemic cardiomyopathy. Protein homeostasis pathways were identified exclusively in PLN-R14∆/∆ 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 amongst the first manifestations of this disease, which could possibly be a novel target for therapy.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Heart

SUBMITTER: Xiaoke Yin

LAB HEAD: Manuel Mayr

PROVIDER: PXD024594 | Pride | 2021-11-03

REPOSITORIES: Pride

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Protein Aggregation Is an Early Manifestation of Phospholamban p.(Arg14del)-Related Cardiomyopathy: Development of PLN-R14del-Related Cardiomyopathy.

Eijgenraam Tim R TR Boogerd Cornelis J CJ Stege Nienke M NM Oliveira Nunes Teixeira Vivian V Dokter Martin M MM Schmidt Lukas E LE Yin Xiaoke X Theofilatos Konstantinos K Mayr Manuel M van der Meer Peter P van Rooij Eva E van der Velden Jolanda J Silljé Herman H W HHW de Boer Rudolf A RA

Circulation. Heart failure 20210930 11

<h4>Background</h4>The p.(Arg14del) pathogenic variant (R14del) of the <i>PLN</i> (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.<h4>Methods</h4>We investigated the progression of cardiomyopathy in PLN-R14<sup>Δ/Δ</sup> mice using echocardiography, ECG, and ...[more]

PMID: 34587756

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Project description: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 (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 PCR 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.

Project description:Background: The phospholamban (PLN) p.Arg14del mutation belongs to the established causes of dilated cardiomyopathy, with the molecular disease mechanisms incompletely understood. We used human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes, CRISPR/Cas9 gene editing and patient heart samples to investigate the molecular pathomechanisms of PLN p.Arg14del cardiomyopathy. Methods and results: Patient dermal fibroblasts carrying the PLN p.Arg14del mutation were reprogrammed into hiPSC, isogenic controls were established by CRISPR/Cas9. Cells were differentiated into cardiomyocytes and characterized in 2D and 3D-engineered heart tissue (EHT) format. Mutant cardiomyocytes revealed significantly prolonged Ca2+ transient decay time, Ca2+-load dependent irregular beating pattern and lower peak force compared to isogenic controls. While this data support the reported super-inhibitory effect of PLN p.Arg14del on the sarcoplasmic/endoplasmic reticulum Ca2+ ATPase, an unchanged SR Ca2+ content argued against disturbed SR Ca2+ cycling as the sole cause of contractile dysfunction. Label-free proteomic analysis of p.Arg14del EHTs revealed less endoplasmic reticulum (ER), ribosomal and mitochondrial proteins. Electron microscopy showed dilation of the rough ER, large lipid droplets in close association with mitochondria and reduced mitochondrial number. Follow-up experiments confirmed impairment of the rough ER/mitochondria compartment and enhanced oxidative stress in PLN p.Arg14del. Relevance for human disease was demonstrated by immunohistochemistry of human heart samples. PLN p.Arg14del end-stage heart failure samples revealed perinuclear aggregates positive for ER marker proteins and oxidative stress in comparison to ischemic heart failure - and non-failing donor heart samples. Surprisingly, transduction of PLN p.Arg14del EHTs with the Ca2+ binding protein GCaMP6f reversed the contractile, molecular and morphological disease phenotype. Conclusion: This study identified impairment of the rough ER/mitochondria compartment without SR dysfunction as a novel disease mechanism underlying PLN p.Arg14del cardiomyopathy. The pathology was improved by Ca2+-scavenging, suggesting impaired local Ca2+ cycling as an important disease culprit.