DWORF Gene Therapy Improves Cardiac Calcium Handling and Mitochondrial Function

Abstract

Background: Calcium (Ca²⁺) dysregulation is a hallmark of heart failure, impairing excitation-contraction coupling and contributing to pathological remodeling. The sarco/endoplasmic reticulum Ca²⁺ ATPase isoform 2a (SERCA2a) mediates Ca²⁺ reuptake into the sarcoplasmic reticulum (SR) during diastole, but its activity declines in failing hearts. DWORF (dwarf open reading frame), a newly identified cardiac microprotein, enhances SERCA2a activity and improves cardiomyocyte Ca²⁺ cycling and contractility. SR Ca²⁺ release also influences mitochondrial metabolism and ATP production. Here, we investigated whether DWORF overexpression improves SR Ca²⁺ handling, augments mitochondrial Ca²⁺ signaling, and protects against heart failure progression.

Methods: Transgenic and adeno-associated virus approaches were used to overexpress DWORF in the heart. Mice underwent transverse aortic constriction (TAC) to model pressure overload-induced heart failure. Cardiac function, mitochondrial metabolism, SR Ca²⁺ uptake, and remodeling were assessed.

Results: Mitochondria from DWORF transgenic hearts displayed increased basal respiration, maximal respiration, and spare respiratory capacity, correlating with enhanced mitochondrial Ca²⁺ uptake kinetics. Western blot analysis showed elevated levels of active PDH (pyruvate dehydrogenase) and mitochondrial Ca²⁺ uniporter expression in DWORF transgenic hearts, supporting a role for DWORF in Ca²⁺-driven metabolic regulation. Similarly, MyoAAV (myo-adeno-associated virus)-mediated DWORF overexpression enhanced mitochondrial respiration and increased levels of active PDH in adult mice. Following TAC, MyoAAV-DWORF-treated mice maintained higher left ventricular function and were protected from further deterioration compared with controls. This benefit was observed when DWORF gene therapy was delivered preventively at the time of pressure overload or after heart failure was already established. DWORF gene therapy also attenuated remodeling, with lower heart weight and lung weight-to-tibia length ratios. Seahorse analysis confirmed sustained mitochondrial improvements in both treatment paradigms.

Conclusions: DWORF overexpression enhances SR Ca²⁺ dynamics, improves mitochondrial energetics, and attenuates pathological remodeling and heart failure progression in response to pressure overload. These findings support DWORF as a promising therapeutic target for heart failure.

Keywords: calcium; genetic therapy; heart failure; micropeptides; mitochondria; myocytes, cardiac.

Figure 1.. DWORF overexpression increases mitochondrial function and enhances active pyruvate dehydrogenase levels in transgenic mice.

A, Oxygen consumption rate (OCR) measurements in cardiac mitochondria isolated from WT and DWORF Tg mice at 2–6 months of age. Arrows denote sequential addition of ADP [0.5mM], oligomycin [1mM], FCCP [4μM], and rotenone [0.5μM]/antimycin A [4μM]. B-D, Quantification of basal respiration (B, measured in presence of ADP), maximal respiration (C, after FCCP addition), and spare respiratory capacity (D, difference in OCR between maximal and basal respiration) from stress test assays shown in panel A. N=10 per group (5 Males/5 Females). E, Western blot analysis of electron transport chain subunits I-V and α-actinin protein expression in heart tissue from mice with the indicated genotypes. F, Quantification of Western blot data in panel E normalized to α-actinin and shown as relative to WT. Oxidative phosphorylation (Ox Phos) representative immunoblots are shown for N=6 mice/group (3 males/3 females). G, Western blot analysis of phospho-PDH (P293), total PDH, PDP1, PDK4 and α-actinin in heart tissue following 36 hours of fasting. H-K, Western blot quantification of phospho-PDH normalized to total PDH (H) and total PDH (I), PDK4 (J), and PDP1 (K) normalized to α-actinin. All data are shown as relative to WT. N=8 mice/group (WT 5 males/3 females; DWORF Tg 7 males/1 female). All data are presented as mean ± SD. Statistical significance was determined using Welch’s t-test (B-D) or Mann-Whitney test (F and H-K). Exact P-values are reported, with statistical significance defined as *P<0.05, **P<0.01.

Figure 2.. DWORF overexpression enhances mitochondrial Ca²⁺ uptake rates and increases MCU expression in transgenic mice.

A, Mitochondrial Ca²⁺ uptake in isolated heart mitochondria from WT and DWORF Tg mice measured using calcium retention capacity (CRC) assays. Calcium Green-5N [250nM] was used as a Ca²⁺ indicator. Arrows indicate sequential addition of 2μL of 10 mM CaCl₂ pulses to 1mL solution. B, Representative traces of mitochondrial swelling recorded during CRC assays. C, Representative single Ca²⁺ uptake trace from the CRC assays shown in panel A. D,E, Quantification of Ca²⁺ decay rates (Tau, D) and initial rate of fall (E) following the addition of extramitochondrial Ca²⁺, which reflects mitochondrial Ca²⁺ uptake kinetics. N=10 mice/group (5 males/5 females). F, Western blot analysis of mitochondrial calcium uniporter (MCU) and α-actinin protein expression in heart tissue from WT and DWORF Tg mice. Representative immunoblots are shown for N=6 mice/group (3 males/3 females). G, Quantification of MCU protein levels normalized to α-actinin. Data are shown as relative to WT for N=6 mice/group (3 males/3 females). All data are presented as mean ± SD. Statistical significance was determined using Mann-Whitney test (D and G) or Welch’s T-test (E). Exact P-values are indicated, with statistical significance defined as *P<0.05, **P<0.01, ***P<0.001.

Figure 3.. Acute DWORF overexpression with MyoAAV enhances mitochondrial respiration and active PDH levels.

A, Oxygen consumption rate (OCR) measurements in cardiac mitochondria isolated from C57BL/6 mice treated with MyoAAV-tdTomato (control) or MyoAAV-DWORF 2 weeks post-viral delivery. Arrows denote sequential addition of ADP [0.5mM], oligomycin [1mM], FCCP [4μM], and rotenone [0.5μM]/antimycin A [4μM]. B-D, Quantification of basal respiration (B, measured in presence of ADP), maximal respiration (C), and spare respiratory capacity (D) from stress test assays shown in panel A. N=6 mice/group (3 males/3 females). E, Western blot analysis of phospho-PDH (P293), total PDH, MCU, tdTomato (red fluorescent protein, RFP), DWORF and α-actinin in heart tissue from mice 2 weeks post-MyoAAV delivery. Representative immunoblots are shown for N=5 tdTomato (2 males/3 females) and N=6 DWORF (3 males/3 females) mice. F-I, Western blot quantification of phospho-PDH normalized to total PDH (F) and total PDH (G), MCU (H), and DWORF (I) normalized to α-actinin. All data are shown as relative to MyoAAV-tdTomato controls. N=5 tdTomato (2 males/3 females), N=6 DWORF (3 males/3 females). All data are presented as means ± SD. Statistical significance was determined using Mann-Whitney test. Exact P-values are indicated, with statistical significance defined as *P<0.05, **P<0.01.

Figure 4.. DWORF gene therapy attenuates heart failure progression and cardiac remodeling following pressure overload injury.

A, Schematic of experimental design and timeline. B,C, Biweekly serial echocardiography analysis of cardiac function in mice from the indicated groups, measured from baseline (pre-sham/TAC) to 12 weeks post-surgery. The analyzed parameters include ejection fraction (B, EF%), and LVID;s (C, left ventricular internal diameter; systole). Bar graphs compare TAC mice at baseline and 12 weeks post-TAC to highlight the effect of MyoAAV-DWORF therapy in mitigating heart failure progression. Data are shown for N=20 tdTomato sham (10 males/10 females), N=20 DWORF sham (10 males/10 females), N=21 tdTomato TAC (13 males/8 females), and N=21 DWORF TAC (11 males/10 females) mice/group. D, Western blot analysis confirming overexpression of tdTomato or DWORF in sham and TAC heart tissue from male and female mice. GAPDH is used as loading control. An overexposed/saturated (Sat.) DWORF immunoblot is included to visualize endogenous baseline DWORF expression. Note that the representative immunoblot shown for GAPDH is the same in panel 4D and 5I, as these are identical samples. E, Quantification of DWORF protein levels in mice from the indicated groups. Data are shown for N=10 tdTomato sham (5 males/5 females), N=10 DWORF sham (5 males/5 females), N=9 tdTomato TAC (5 males/4 females), and N=11 DWORF TAC (6 males/5 females) mice/group. DWORF expression is normalized to GAPDH, and all data are shown as relative to sham mice treated with MyoAAV-tdTomato. F, Representative images whole hearts (top) and Picrosirius red stained heart sections (bottom) from sham and TAC mice. G,H, Heart weight (G) and lung weight (H) normalized to tibia length (mg/mm) in sham and TAC MyoAAV-tdTomato and MyoAAV-DWORF groups 12 weeks post-surgery. N=20 tdTomato sham (10 males/10 females), N=20 DWORF sham (10 males/10 females), N=16 tdTomato TAC (8 males/8 females), and N=18 DWORF TAC (8 males/10 females) mice/group. I, Quantification of percentage of fibrotic area by Picrosirius red staining. N=11 tdTomato sham (6 males/5 females), N=11 DWORF sham (6 males/5 females), N=13 tdTomato TAC (9 males/4 females), N=14 DWORF TAC (9 males/5 females) mice/group. Statistical significance was analyzed using two-way ANOVA with Geisser-Greenhouse correction and Tukey’s multiple comparison test (B, C), Kruskal-Wallis test with Dunn’s multiple comparison test (E, H, I), or Welch’s t-test (G). Exact P-values are indicated, with significance defined as *P<0.05, **P<0.01, ***P<0.001, and ****P<0.0001 for MyoAAV-DWORF TAC vs. MyoAAV-tdTomato TAC mice. For all data shown, values are represented as circles (males) and triangles (females), and data are expressed as mean ± SD.

Figure 5.. MyoAAV-mediated DWORF overexpression enhances mitochondrial respiration and SR Ca²⁺ dynamics following sham and TAC procedures.

A, Oxygen consumption rate (OCR) measurements in isolated heart mitochondria from MyoAAV-tdTomato and MyoAAV-DWORF mice 12 weeks post-sham surgery. Arrows denote sequential addition of ADP [0.5mM], oligomycin [1mM], FCCP [4μM], and rotenone [0.5μM]/antimycin A [4μM]. B–D, Quantification of mitochondrial respiration parameters in sham mice including basal respiration (B, OCR after ADP addition), maximal respiration (C, OCR after FCCP addition), and spare respiratory capacity (D, difference in OCR between maximal and basal respiration). N=13 tdTomato sham (8 males/5 females) and N=14 DWORF sham (9 males/5 females) mice/group. E, OCR measurements in isolated heart mitochondria from MyoAAV-tdTomato and MyoAAV-DWORF mice 12 weeks post-TAC surgery. F–H, Quantification of mitochondrial respiration parameters in TAC mice including basal respiration (F), maximal respiration (G), and spare respiratory capacity (H) for N=15 tdTomato TAC (10 males/5 females) and N=17 DWORF TAC (12 males/5 females) mice/group. I, Western blot analysis of phospho-PDH (P293), total PDH, and GAPDH (loading control) in heart tissue 12 weeks post-sham or TAC surgery. Note that the representative immunoblot shown for GAPDH is the same in panel 4D and 5I, as these are identical samples J,K, Western blot quantification of phospho-PDH normalized to total PDH (J) and total PDH normalized to GAPDH (K) for N=9 tdTomato sham (5 males/4 females), N=10 DWORF sham (5 males/5 females), N=9 tdTomato TAC (5 males/4 females), and N=11 DWORF TAC (6 males/5 females) mice/group. Data are shown as relative to control mice (sham+MyoAAV-tdTomato). L-N, IonOptix analysis of paced isolated cardiomyocytes from sham and TAC MyoAAV-tdTomato and MyoAAV-DWORF groups. Parameters measured include cytosolic Ca²⁺ decay rates (Tau, L) and cardiomyocyte fractional shortening (M). Representative calcium transients (Top) and sarcomere length (bottom) traces are shown in panel N. Violin plots show individual cells with 104 cells from tdTomato sham (N=9, 4 males and 5 females), 88 cells from DWORF sham (N=9, 4 males and 5 females), 135 cells from tdTomato TAC (N=8, 4 males and 4 females), and 135 cells from DWORF TAC (N=9, 4 males and 5 females). Statistical significance was determined using Welch’s T-test (B and F), Mann-Whitney test (C, D, G, and H), Kruskal-Wallis test and Dunn’s multiple comparison test (J and K), or using hierarchical analysis using a linear mixed-effects model with Holm-Šídák post hoc correction (L and M). Exact P-values are indicated, with statistical significance defined as *P<0.05, **P<0.01, ***P<0.001. For all data shown, values are represented as circles (males) and triangles (females), and data are expressed as mean ± SD.

Figure 6.. DWORF gene therapy stabilizes cardiac function when administered after heart failure onset.

A, Schematic of experimental design and timeline. B-C, Biweekly serial echocardiography analysis of cardiac function in mice from the indicated groups measured from baseline (pre-TAC) to 16 weeks post-TAC. The analyzed parameters include ejection fraction (B, EF%), and LVID;s (C, left ventricular internal diameter; systole). Bar graphs compare TAC mice at baseline, 6-, and 12-weeks post-TAC to highlight the effect of MyoAAV-DWORF therapy in mitigating heart failure progression after delivery. Data are shown for N=24 tdTomato TAC (10 males/14 females), and N=27 DWORF TAC (13 males/14 females) mice/group. D, Western blot analysis of tdTomato or DWORF in 16 weeks post-TAC heart tissue from male and female mice. GAPDH is used as loading control. Note that the representative immunoblot shown for GAPDH is the same in panel 6D and 7E, as these are identical samples. E, Quantification of DWORF protein levels in mice from the indicated groups. Data are shown for N=10 tdTomato (5 males/5 females), and N=12 DWORF (6 males/6 females) mice/group. DWORF expression is normalized to GAPDH, and all data are shown as relative to MyoAAV-tdTomato treated mice. F, Representative images of whole hearts (top), and Picrosirius red stained heart sections (bottom) from 16-week-TAC mice. G, Quantification of percentage of fibrotic area by Picrosirius red staining. N=12 tdTomato TAC (4 males/8 females), N=18 DWORF TAC (8 males/10 females) mice/group. H, Heart weight normalized to tibia length (mg/mm) in MyoAAV-tdTomato and MyoAAV-DWORF groups 16 weeks post-surgery. N=23 tdTomato TAC (9 males/14 females), and N=25 DWORF TAC (11 males/14 females) mice/group. I, Lung weight normalized to tibia length (mg/mm) in MyoAAV-tdTomato and MyoAAV-DWORF groups 16 weeks post-surgery. N=22 tdTomato TAC (8 males/14 females), and N=23 DWORF TAC (9 males/14 females) mice/group. Statistical significance was analyzed using two-way ANOVA with Geisser-Greenhouse correction and Fisher’s least significant difference comparison test (B,C), Welch’s t-test (E), or Mann-Whitney test (G-I). Exact P-values are indicated, with significance defined as *P<0.05, **P<0.01, and ****P<0.0001 for MyoAAV-DWORF vs. MyoAAV-tdTomato mice. For all data shown, values are represented as circles (males) and triangles (females), and data are expressed as mean ± SD.

Figure 7.. MyoAAV-mediated DWORF overexpression enhances mitochondrial respiration and SR Ca²⁺ dynamics when administered after established heart failure.

A, Oxygen consumption rate (OCR) measurements in isolated heart mitochondria from MyoAAV-tdTomato and MyoAAV-DWORF mice 16 weeks post-TAC surgery. Arrows denote sequential addition of ADP [0.5mM], oligomycin [1mM], FCCP [4μM], and rotenone [0.5μM]/antimycin A [4μM]. B–D, Quantification of mitochondrial respiration parameters in 16-week post-TAC mice including basal respiration (B, OCR after ADP addition), maximal respiration (C, OCR after FCCP addition), and spare respiratory capacity (D, difference in OCR between maximal and basal respiration). N=13 tdTomato (5 males/8 females) and N=15 DWORF (7 males/8 females) mice/group. E, Western blot analysis of phospho-PDH (P293), total PDH, and GAPDH (loading control) in heart tissue 16 weeks post-TAC. Note that the representative immunoblot shown for GAPDH is the same in panel 6D and 7E, as these are identical samples. F,G, Western blot quantification of phospho-PDH normalized to total PDH (F) and total PDH normalized to GAPDH (G) for N=10 tdTomato (5 males/5 females), N=12 DWORF (6 males/6 females) mice/group. Data are shown as relative to control mice (TAC+MyoAAV-tdTomato). H-J, IonOptix analysis of paced isolated cardiomyocytes (CM) from TAC MyoAAV-tdTomato and MyoAAV-DWORF groups. Parameters measured include cytosolic Ca²⁺ decay rates (Tau, H), cardiomyocyte fractional shortening (I), and representative Fura-2 (Top) and sarcomere length (bottom) traces (J) Violin plots show individual cells with 98 cells from tdTomato TAC (N=10, 4 males and 6 females), and 114 cells from DWORF TAC (N=11, 4 males and 7 females). Statistical significance was determined using Welch’s T-test (B-D and G), Mann-Whitney test (F), or using hierarchical analysis using a linear mixed-effects model followed by Holm-Šídák post hoc correction (H and I). Exact P-values are indicated, with statistical significance defined as *P<0.05, **P<0.01, ****P<0.0001. For all data shown, values are represented as circles (males) and triangles (females), and data are expressed as mean ± SD.