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, R14Δ/Δ (homozygous PLN-R14del) mice carrying the DWORF transgene (R14Δ/ΔDWORFTg [R14Δ/Δ mice carrying the DWORF transgene]) were used. RESULTS: DWORF expression was suppressed in hearts of R14Δ/Δ mice with severe heart failure. Restoration of DWORF expression in R14Δ/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 R14Δ/Δ cardiomyocytes, sarcoplasmic reticulum calcium reuptake and relaxation were already enhanced, and no differences were detected between R14Δ/Δ and R14Δ/ΔDWORFTg. 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 R14Δ/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 R14Δ/Δ mice, by reducing abnormal S/ER clusters.</p