Gene editing reverses arrhythmia susceptibility in humanized PLN-R14del mice: modelling a European cardiomyopathy with global impact

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.

Keywords: CRISPR/Cas9; Gene therapy; Humanized mouse; Phospholamban R14del mutation; Ventricular tachycardia.

Graphical Abstract

Figure 1

Generation of knock-in humanized PLN-R14del mouse model. (A) Schematic of the targeting construct. A LoxP-H2B-GFP-4XpolyA-FRT-Neo-FRT-LoxP-hPLN^WT/R14del cassette was inserted into mouse PLN exon 2. (B) PCR analysis of genomic DNA from targeted embryonic stem cells. (C) Genotyping by tail PCR and (D) Sanger sequencing method.

Figure 2

Histological analysis and cardiac MRI. Masson’s trichrome (A) and haematoxylin and eosin (H&E) staining (B) of heart sections from hPLN-WT and hPLN-R14del mice. Scale bars, 100 μm for panel A, 1 mm for panel B. (C) Bar graph showing right ventricular end-diastolic volume (RVED), left ventricular end-diastolic volume (LVEDV), and right and left ventricular stroke volume (RVSV and LVSV) measured by cardiac MRI (n = 6 per group; unpaired two-tailed Student’s t-test. *P < 0.05).

Figure 3

hPLN-R14del hearts showed increased risk of rapid pacing-induced arrhythmia. (A) Rapid pacing of isolated mouse hearts during adrenergic stimulation with isoproterenol (ISO) readily induced sustained ventricular tachycardia (VT) in hPLN-R14del group (bottom), whereas conversion to sinus rhythm was more prevalent in the hPLN-WT group (top), based on volumetric electrocardiogram (vECG) recordings. (B) the majority of hPLN-R14del (6/8) but not hPLN-WT (3/11) hearts exhibited sustained VT in response to challenge up to predefined cut-off frequency of 22.5 Hz. (C) A significant decrease in VT threshold was observed in the hPLN-R14del group compared to hPLN-WT (hPLN-WT: n = 11; hPLN-R14del: n = 8; unpaired two-tailed Student’s t-test. *P < 0.05).

Figure 4

Proteomic analysis of right and left ventricles from hPLN-WT and hPLN-R14del mice. (A) Experimental design. (B) Volcano plots showing up-regulated (yellow, LV; red, RV) and down-regulated proteins (cyan, LV; dark blue, RV) in left ventricle (left panel) and right ventricle (right panel) based on proteomic analysis of the indicated samples (n = 6 per group). (C) Up- and down-regulated proteins in the left and right ventricle were subjected to dynamic enrichment analysis using the Molecular Biology of the Cell Ontology (MBCO), and disease relevant networks are shown. Arrows connect level-2 parent subcellular processes (SCPs) with their level-3 child SCPs, and SCPs with their annotated differentially expressed proteins. All proteins annotated to level-3 child SCPs are also annotated to their level-2 parent SCPs. For simplicity, we only show the annotation with level-3 child SCPs in these cases. Dashed lines connect functionally related SCPs of the same level, as predicted by the MBCO algorithm. Frames enclose SCP networks that converge on the indicated biological mechanism.

Figure 5

In vivo CRISPR-Cas9 gene editing efficiency. (A) Primers and probes (FAM and HEX) for NHEJ edit detection assays. (B) Allelic discrimination by ddPCR. Frequency of indels at on-target (R14del) site in genomic DNA from both ventricular tissues (C) and isolated cardiomyocytes (D) in CRISPR-treated vs. Control mice (ventricular tissues from controls: n = 6 and CRISPR-treated: n = 7; isolated cardiomyocytes from controls and CRISPR-treated: n = 3; unpaired two-tailed Student’s t-test. *P < 0.05).

Figure 6

Effect of CRISPR treatment on cardiac function. CRISPR treatment significantly reduced ventricular end-diastolic and stroke volumes (A). (hPLN-WT: n = 6; hPLN-R14del: n = 6; CRISPR-treated: n = 5; one-way ANOVA, Tukey’s multiple comparison test: *P < 0.05, **P < 0.01 vs. hPLN-WT, ^§P < 0.05 vs. hPLN-R14del). Greater than two-fold reduction of pacing induced arrhythmia by chosen cut-off of 22.5Hz (B) and a significant increase in VT threshold (C) revealed prominent decreased risk of pacing induced arrhythmia in the CRISPR-treated hPLN-R14del group (Controls: n = 6; CRISPR-treated: n = 8; unpaired two-tailed Student’s t-test. **P < 0.01).