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

Abstract

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 ∆/∆ 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 ∆/∆ 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 ∆/∆ 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.

Antisense Therapy Attenuates Phospholamban p.(Arg14del) Cardiomyopathy in Mice and Reverses Protein Aggregation

“…To investigate whether administration of a PLN-ASO could halt or even reverse established PLN-R14del cardiomyopathy, we aimed to initiate treatment when PLN-R14 Δ/Δ mice had considerable HF. Based on previously published data on the development of DCM in PLN-R14 Δ/Δ mice [ 15 , 16 ], we decided to start PLN-ASO injections at 5 (ASO-early) or 6 (ASO-late) weeks of age when mice were shown to exhibit moderate (27.5 ± 0.2% fractional shortening (FS) vs. 36.2 ± 1.1% in age-matched wild-type (WT) controls) or severe (13.6 ± 2.3% FS) LV dysfunction with ventricular dilatation, respectively ( Figure 1 A and Figure S1 ). As expected [ 16 ], vehicle-treated PLN-R14 Δ/Δ mice reached a humane endpoint at 8.1 ± 0.5 weeks of age ( Figure 1 B).…”

“…We have previously reported that, like in human patients [ 10 , 24 ], intracardiomyocyte aggregation of PLN proteins is an early hallmark of PLN-R14del cardiomyopathy in mice [ 15 ]. To determine PLN protein distribution, we performed immunofluorescent staining for PLN on cardiac sections.…”

“…We have recently characterized the PLN-R14del mouse model, and have described that PLN-R14 Δ/Δ mice rapidly and progressively develop cardiomyopathy with severe HF between 3 and 8 weeks of age [ 15 , 16 ]. In addition, pre-emptive depletion of PLN using a PLN-ASO demonstrated beneficial effects in three murine models of heart disease [ 18 ].…”

“…Interestingly, the PLN-R14del pathogenic variant is characterized by dense perinuclear protein aggregation [ 10 ], a phenomenon shared with several other pathogenic DCM and ACM gene variants [ 11 , 12 , 13 ]. Since protein aggregation is a well-known pathogenic hallmark of many diseases and in aging [ 14 ], PLN protein aggregation has been implicated as a potential mechanism driving cardiac remodeling in PLN-R14del cardiomyopathy [ 10 , 15 ]. We have recently developed a mouse model carrying the PLN-R14del pathogenic variant, which recapitulates most disease characteristics that are observed in human patients [ 16 ].…”

“…We have recently developed a mouse model carrying the PLN-R14del pathogenic variant, which recapitulates most disease characteristics that are observed in human patients [ 16 ]. Mice that are homozygous for the PLN-R14del pathogenic variant (PLN-R14 Δ/Δ ) develop cardiomyopathy, PLN protein aggregation and HF in an accelerated manner with early mortality within 2 months, providing an opportunity to screen novel therapies [ 15 , 16 ].…”