PMC

(A) Ca²⁺-dependent Ca²⁺-uptake assays were performed using total homogenates from hearts of WT, αMHC-DWORF (Tg), and Dworf KO mice to directly measure SERCA affinity for Ca²⁺ (K_Ca) and SERCA activity. Mean K_Ca values from n = 8 hearts of each genotype (bar graphs). P < 0.05. (B) Ca²⁺-dependent Ca²⁺-uptake assays were performed using total homogenates from soleus muscles of WT and Dworf KO mice. Mean K_Ca values from mice of each genotype (bar graphs). P < 0.05, n = 8. (C) Working model for DWORF function.

(A) Two-photon scanning confocal microscopy of the flexor digitorum brevis muscle of adult mice after in vivo electroporation of plasmids encoding GFP-DWORF, GFP-PLN, or GFP-SLN indicates that DWORF localization closely resembles that of SR proteins PLN and SLN (M, M-line; Z, Z-line; scale bar, 5 μm). (B) Colocalization of GFP-DWORFand mCherry-SERCA in transfected COS7 cells (scale bar, 5 μm). (C) Coimmunoprecipitation experiments in transfected COS7 cells using GFP-DWORFand Myc-tagged SERCA isoforms. IP, immunoprecipitation. (D) Immunoprecipitation of Myc-SERCA from lysates of COS7 cells transfected with equal amounts of HA-DWORF, -PLN, -SLN, or -MLN and Myc-SERCA with fivefold overexpression of either GFP or GFP-DWORF. Coexpression of GFP-DWORF reduced the pull-down of HA-tagged peptides in association with SERCA, which indicated that DWORF binding to SERCA excludes binding of PLN, SLN, or MLN.

(A) Northern blot of adult mouse tissues showing Dworf RNA expression. (B) Western blot of adult mouse tissues with the DWORF-specific antibody reveals a single band at the predicted size of 3.8 kD. Quad, quadriceps; G/P, gastrocnemius/plantaris;TA, tibialis anterior; EDL, extensor digitorum longus. (C) Detection of Dworf RNA by qRT-PCR in 6-month-old WT and αMHC-CnA mice. Mean ± SEM; WT, n = 4; Tg, n = 5. (D) Western blot analysis of heart homogenates from WT and αMHC-calcineurin mice immunoblotted with DWORF-specific antibody. (E) qRT-PCR analysis of human ischemic heart failure tissue showing reduced DWORF mRNA in failing hearts, whereas atrial natriuretic peptide (NPPA) is significantly increased. Means ± SEM; nonfailing, n = 8; failing, n = 8.

(A) A CRISPR gRNA was generated to target the coding sequence of exon 2. An allele containing a 2-bp insertion was chosen for further experiments. The mutation is expected to produce a truncated protein lacking the transmembrane domain. (B) Western blot showing the absence of DWORF protein in the cardiac ventricle and soleus muscle of Dworf KO mice. (C) Representative Ca²⁺ transients and SR load measurements recorded in fluo-4–loaded cardiomyocytes from WT, αMHC-DWORF (Tg), and Dworf KO mice. (D) Mean amplitude of pacing-induced Ca²⁺ transients in fluo-4–loaded cardiomyocytes from WT, Tg, and KO mice and caffeine-induced Ca²⁺ transients triggered by rapid application of 10 mM caffeine to quantify SR load. Ca²⁺ signal is shown as fluorescence ratio (F/F₀) with the fluorescence intensity (F) normalized to the minimal intensity measured between 0.5 Hz contractions at diastolic phase (F₀). P < 0.05, n = 6. (E) Average decay-time constants (Tau) of pacing-induced Ca²⁺ transients in WT, Tg, and Dworf KO cardiomyocytes measured by fitting a single exponential to the Ca²⁺ transient decay trace.This parameter is indicative of SERCA activity. P < 0.05, n = 8. (F) Isometric force was measured from soleus muscles mounted ex vivo and stimulated by 0.2-ms current pulses applied at a range of frequencies. (Left) Force decay was slower in Dworf KO muscles (arrow) after fully fused tetanic contractions as shown for 90 Hz (inset). (Right) Slower relaxation for Dworf KO muscles occurred for stimulus frequencies sufficient to produce twitch fusion (>20 Hz); however, unfused twitches at low frequency showed no difference in relaxation rates. P < 0.05, n = 6.