Abstract GS.13: Dworf Overexpression In The Heart Results In Enhanced Mitochondrial Function

Objective: Previously, we identified dwarf open reading frame (DWORF) as a microprotein that regulates SERCA2a in the sarcoplasmic reticulum (SR). New data from our lab indicates that a fraction of DWORF localizes to the mitochondria. The goal of these studies was to analyze the role of DWORF in the mitochondria. Methods & Results: To identify novel DWORF interacting proteins, we performed a BioID assay in neonatal rat ventricular myocytes (NRVMs) expressing a mTurbo-DWORF fusion protein. In addition to identifying SR protein SERCA2a, we unexpectedly identified several mitochondrial proteins. Percoll gradient fractionation of mouse heart lysates showed DWORF is distributed across SR, mitochondria associated membranes (MAMs), and purified mitochondrial fractions. Seahorse mito stress test performed in isolated mitochondria from wildtype (WT), DWORF transgenic (Tg), DWORF knockout (KO), and PLN KO mouse hearts showed that mitochondria from DWORF Tg mice have increased maximal O 2 consumption rates (OCR) while mitochondria from DWORF KO hearts have reduced OCR compared to WT. To measure mitochondrial Ca 2+ dynamics, we performed mitochondrial Ca 2+ uptake assays and found that DWORF Tg hearts have increased Ca 2+ uptake rates while DWORF KO mice have reduced Ca 2+ uptake rates compared to WT. Western blot (WB) analysis of mito extracts from WT and DWORF Tg hearts indicated elevated levels of the mitochondrial Ca 2+ uniporter (MCU), while no observable changes were detected in electron transport chain (ETC) subunit expression. WB analysis also revealed a reduction in phosphorylated pyruvate dehydrogenase (PDH) in DWORF Tg mice, indicating that the enhanced mitochondrial activity observed in DWORF Tg mice may be due to a more active TCA cycle. Additionally, metabolomics analysis shows that DWORF Tg mice have lower levels of fatty acids and carnitines than WT. Conclusions: DWORF overexpression leads to increased mitochondrial respiration, enhanced mitochondrial Ca 2+ uptake, and reduced PDH phosphorylation (increased PDH activity), while DWORF loss-of-function results in the opposite phenotype. Collectively, these data indicate that DWORF plays a previously unrecognized role in the mitochondria and contributes to mito Ca 2+ regulation and energetics.