DWORF

The phospholamban (PLN) pathogenic gene variant p.Arg14del causes cardiomyopathy, which is characterized by perinuclear PLN protein clustering and can lead to severe heart failure (HF). Elevated expression of Dwarf open reading frame (DWORF), a protein counteracting the function of PLN in the sarcoplasmic reticulum (SR), can delay disease progression in a PLN-R14del mouse model. Here, we evaluated whether deletion of DWORF (DWORF^-/-) would have an opposite effect and accelerate age-dependent disease progression in wild-type (WT) mice and mice with a pathogenic PLN-R14del allele (R14^Δ/+). We show that DWORF^-/- mice maintained a normal left-ventricular ejection fraction (LVEF) during ageing and no difference with WT control mice could be observed up to 20 months of age. R14^Δ/+ mice maintained a normal cardiac function until 12 months of age, but at 18 months of age LVEF was significantly reduced as compared with WT mice. Absence of DWORF did not accelerate the R14^Δ/+-induced reduction in LVEF, nor enhance the increases in gene expression of markers related to cardiac remodeling and fibrosis and did not exacerbate cardiac fibrosis caused by the R14^Δ/+ mutation. Together, these results demonstrate that absence of DWORF does not accelerate nor exacerbate PLN-R14del cardiomyopathy in mice harboring the pathogenic R14del allele. In addition, our data indicate that DWORF appears to be dispensable for cardiac function during ageing.

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.

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/ΔDWORF^Tg [R14^Δ/Δ mice with a copy of the DWORF transgene]) were used.

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/ΔDWORF^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.

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.

Background Cardiomyopathy is a leading health threat in Duchenne muscular dystrophy (DMD). Cytosolic calcium upregulation is implicated in DMD cardiomyopathy. Calcium is primarily removed from the cytosol by the sarcoendoplasmic reticulum calcium ATPase (SERCA). SERCA activity is reduced in DMD. Improving SERCA function may treat DMD cardiomyopathy. Dwarf open reading frame (DWORF) is a recently discovered positive regulator for SERCA, hence, a potential therapeutic target. Methods and Results To study DWORF's involvement in DMD cardiomyopathy, we quantified DWORF expression in the heart of wild-type mice and the mdx model of DMD. To test DWORF gene therapy, we engineered and characterized an adeno-associated virus serotype 9-DWORF vector. To determine if this vector can mitigate DMD cardiomyopathy, we delivered it to 6-week-old mdx mice (6×10¹² vector genome particles/mouse) via the tail vein. Exercise capacity, heart histology, and cardiac function were examined at 18 months of age. We found DWORF expression was significantly reduced at the transcript and protein levels in mdx mice. Adeno-associated virus serotype 9-DWORF vector significantly enhanced SERCA activity. Systemic adeno-associated virus serotype 9-DWORF therapy reduced myocardial fibrosis and improved treadmill running, electrocardiography, and heart hemodynamics. Conclusions Our data suggest that DWORF deficiency contributes to SERCA dysfunction in mdx mice and that DWORF gene therapy holds promise to treat DMD cardiomyopathy.