DWORF Extends Life Span in a PLN-R14del Cardiomyopathy Mouse Model by Reducing Abnormal Sarcoplasmic Reticulum Clusters

Abstract

BACKGROUND: The p.Arg14del variant of the PLN (phospholamban) gene causes cardiomyopathy, leading to severe heart failure. Calcium handling defects and perinuclear PLN aggregation have both been suggested as pathological drivers of this disease. Dwarf open reading frame (DWORF) has been shown to counteract PLN regulatory calcium handling function in the sarco/endoplasmic reticulum (S/ER). Here, we investigated the potential disease-modulating action of DWORF in this cardiomyopathy and its effects on calcium handling and PLN aggregation. METHODS: We studied a PLN-R14del mouse model, which develops cardiomyopathy with similar characteristics as human patients, and explored whether cardiac DWORF overexpression could delay cardiac deterioration. To this end, R14 Δ/Δ (homozygous PLN-R14del) mice carrying the DWORF transgene (R14 Δ/Δ DWORF Tg [R14 Δ/Δ mice with a copy of the DWORF transgene]) were used. RESULTS: DWORF expression was suppressed in hearts of R14 Δ/Δ mice with severe heart failure. Restoration of DWORF expression in R14 Δ/Δ mice delayed cardiac fibrosis and heart failure and increased life span >2-fold (from 8 to 18 weeks). DWORF accelerated sarcoplasmic reticulum calcium reuptake and relaxation in isolated cardiomyocytes with wild-type PLN, but in R14 Δ/Δ cardiomyocytes, sarcoplasmic reticulum calcium reuptake and relaxation were already enhanced, and no differences were detected between R14 Δ/Δ and R14 Δ/Δ DWORF Tg . Rather, DWORF overexpression delayed the appearance and formation of large pathogenic perinuclear PLN clusters. Careful examination revealed colocalization of sarcoplasmic reticulum markers with these PLN clusters in both R14 Δ/Δ mice and human p.Arg14del PLN heart tissue, and hence these previously termed aggregates are comprised of abnormal organized S/ER. This abnormal S/ER organization in PLN-R14del cardiomyopathy contributes to cardiomyocyte cell loss and replacement fibrosis, consequently resulting in cardiac dysfunction. CONCLUSIONS: Disorganized S/ER is a major characteristic of PLN-R14del cardiomyopathy in humans and mice and results in cardiomyocyte death. DWORF overexpression delayed PLN-R14del cardiomyopathy progression and extended life span in R14 Δ/Δ mice, by reducing abnormal S/ER clusters.

Early consequences of the phospholamban mutation PLN‐R14del+/− in a transgenic mouse model

“…This, and the likely involvement of pathogenetic mechanisms beyond SERCA2a dysregulation, may suggest to invest on mutation reversal, 35 or at least on improvement of mutant protein processing (e.g. by “chaperone” molecules), or reducing the impact of pathogenic PLN protein 13,36 as the most logical mechanism‐based therapeutic approaches. Speaking of more generic approaches, countering the enhancement of sustained Na + current (a common response to cell stress) has proven effective in preventing the consequences of chronic upregulation of Ca 2+ cycling in hiPS‐CMs 22 …”

Unbiased complexome profiling and global proteomics analysis reveals mitochondrial impairment and potential changes at the intercalated disk in presymptomatic R14Δ/+mice hearts

“…Previous reports observed a decrease in conduc on velocity in 12-16wk-old R14 Δ/+ mice hearts 7,39 , which may contribute to the R14Δ-ACM phenotype. Due to the complete absence of fibrosis at this age 7,11 , these conduc on abnormali es have been ini ally a ributed to altered Ca 2+ -homeostasis 7 . ICDs mediate electromechanical coupling between adjacent cardiomyocytes 109 , macrophages 82 and fibroblasts 84,85 .…”

“…A cri cal component of our work is the observa on of disrupted ICD and mitochondrial supercomplexes in juvenile (9wk-old) R14 Δ/+ mice, significantly prior to the onset of cardiomyopathy at 18 months of age 7,11 . Targe ng these pathways at a very young age may represent an effec ve therapeu c strategy that can be delivered prior to the onset of clinical symptoms.…”

“…SEC-DIA-MS/PERCOM analysis was performed on 28wk-old wild-type (WT) and heterozygous R14 Δ/+ mice 7 . These animals, while exhibi ng a mild cardiac conduc on defect 7,39 , lack the severe cardiomyopathy, fibrosis and perinuclear cluster forma on observed in end-stage human-pa ent hearts and thus reflect an early (presymptoma c) disease state 7,11 . Out of 3055 detected proteins, we iden fied 296 with altered elu on profiles in R14 Δ/+ mice.…”

“…On the molecular level, R14Δ-PLN is associated with loss of PKA binding, resul ng in PLN hypo-phosphoryla on leading to altered SERCA2a regula on and Ca 2+ -handling dysfunc on 5,9,10 . On the cellular level, R14∆-PLN is associated with a complex phenotype involving accelerated Ca 2+ -handling dynamics, forma on of PLN-posi ve perinuclear SER clusters, upregulated UPR signaling and mitochondrial dysregula on 7,[11][12][13][14] . Despite over a decade of work, the mechanisms linking R14Δ-PLN to disease pathology remain poorly understood.Many proteins assemble into mul meric and/or mul -protein complexes with well-defined molecular composi on and func on 15,16 .…”