Calcium dysregulation is a universal characteristic of heart failure (HF) and reduced SERCA (sarco/endoplasmic reticulum calcium-ATPase) activity plays a central role in disease progression. 1 Hence, increasing SERCA activity has been pursued as a clinical approach for treating HF and significant evidence supports its therapeutic potential. 1, 2 Recently, we discovered the novel muscle-specific micropeptide dwarf open reading frame (DWORF), which enhances SERCA activity by displacing the SERCA-inhibitory peptide phospholamban. 3, 4 In mice, cardiac-specific transgenic overexpression of DWORF enhanced SERCA activity, increased calcium cycling and contractility, 4 and rescued a genetic model of dilated cardiomyopathy, 3 suggesting that DWORF overexpression might be used to achieve SERCA activation in HF to normalize calcium homeostasis and prevent disease progression.

To explore the therapeutic potential of DWORF gene therapy in HF, we developed an adeno-associated virus (AAV) approach using cardiotropic serotype 9 (AAV9) and the cTnT (cardiac troponin-T) promoter (Addgene plasmid no. 69915). 5 All animal procedures were conducted in accordance with institutional guidelines and were approved by the Institutional Animal Care and Use Committee (IACUC). Both male and female mice were used. AAV9-cTnT-DWORF (AAV-DWORF) and control AAV9-cTnT-tdTomato (AAV-tdTomato) were validated in mice by delivery at postnatal day 5 by intraperitoneal injection at 5× 1013 viral genomes/kg and Western blot analysis revealed cardiac-specific overexpression at postnatal day 28 (Figure [A]). The efficacy of AAV-DWORF gene therapy was assessed in a mouse model of dilated cardiomyopathy caused by gene deletion of muscle-specific