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

Abstract

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.

Aberrant PLN-R14del Protein Interactions Intensify SERCA2a Inhibition, Driving Impaired Ca2+ Handling and Arrhythmogenesis

“…Based on initial findings from PLN-R14del overexpression in a heterologous cell culture system as well as in the mouse heart, PLN-R14del was proposed to exert super-inhibitory effects on SERCA2a activity, leading to cardiac remodeling and early death [5]. Additional evidence has recently emerged from numerous studies on human patients and/or patient-derived cardiomyocytes (iPSC-CMs), and various PLN-R14del animal models [12][13][14][15][16][17][18]. These studies have revealed multiple defects associated with the PLN-R14del mutation, including impaired Ca 2+ homeostasis [12][13][14]16,18], electrical remodeling [19], unfolded protein response (UPR) activation [20], and protein aggregation [15,21,22].…”

“…Additional evidence has recently emerged from numerous studies on human patients and/or patient-derived cardiomyocytes (iPSC-CMs), and various PLN-R14del animal models [12][13][14][15][16][17][18]. These studies have revealed multiple defects associated with the PLN-R14del mutation, including impaired Ca 2+ homeostasis [12][13][14]16,18], electrical remodeling [19], unfolded protein response (UPR) activation [20], and protein aggregation [15,21,22].…”

Phospholamban R14del disease: The past, the present and the future

“…Along those lines, treatment with the small molecule istaroxime that stimulates SERCA2a activity had beneficial effects in the PLN-R14del zebrafish model as it An alternative gene targeting approach includes the use of antisense oligonucleotides (ASO), which bind to a specific mRNA target resulting in mRNA degradation and enable elimination of a mutant protein without the need for genetic modifications (84). The applicability of this approach in the setting of PLN-R14del pathology was recently investigated in the mouse PLN-R14del knock-in model (82,83). Administration of a Pln-targeting ASO in homozygous PLN-R14del mice prevented PLN aggregation, cardiac remodeling and cardiac dysfunction, leading to a 3-fold increase in survival rate (83).…”

“…While it is still unknown what triggers PLN aggregate formation, structural alterations of PLN-R14del may cause protein misfolding that could promote its aggregation. The presence of autophagy marker proteins such as p62 and LC3 within PLN aggregates, along with alterations in their expression levels, indicates inefficient protein degradation by the autophagy pathway and depressed proteostasis ( 48 , 82 ). This defect, along with Ca 2+ mishandling, may be directly or indirectly implicated in activation of compensatory mechanisms such as UPR in order to alleviate ER stress and maintain proteostasis.…”

“…Administration of a Pln -targeting ASO in homozygous PLN-R14del mice prevented PLN aggregation, cardiac remodeling and cardiac dysfunction, leading to a 3-fold increase in survival rate ( 83 ). Importantly, beneficial effects were observed even when ASO were administered after disease onset, as ASO treatment prevented disease progression, but could not reverse existing cardiac tissue damage ( 82 ). While these are encouraging findings and unveil promising avenues for future studies, it is currently unclear whether such a strategy would actually be applicable in human patients due to inherent differences between mouse and human cardiac physiology, as well as Ca 2+ cycling mechanisms.…”