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Transcriptomic analyses of cardiomyopathy development in mice carrying the phospholamban p.(Arg14del) pathogenic variant, and comparison with ischemic heart disease

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 (mild cardiomyopathy), 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 electrocardiogram 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 remodeling, inflammation and metabolic dysfunction, in part similar to ischemic cardiomyopathy. Altered 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.

ORGANISM(S): Mus musculus

PROVIDER: GSE168610 | GEO | 2021/07/22

REPOSITORIES: GEO

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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.

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Transcriptomic analyses of cardiomyopathy development in mice carrying the phospholamban p.(Arg14del) pathogenic variant, and comparison with ischemic heart disease

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