Neddylation is required for perinatal cardiac development through stimulation of metabolic maturation

**Background:** Cardiac maturation after birth involves profound structural and metabolic changes, including a shift from glycolysis to fatty acid oxidation. Disruption of this process can lead to heart failure. While transcriptional regulators of this metabolic switch are known, the role of post-translational modifications like neddylation—the conjugation of NEDD8 to target proteins—remains unclear. Neddylation is known to regulate cullin-RING ubiquitin ligases and has been implicated in diverse cellular processes, but its role in postnatal cardiac development had not been explored.

**Methods:** The authors generated conditional knockout mice lacking NAE1, the catalytic subunit of the NEDD8-activating E1 enzyme, specifically in cardiomyocytes. Because global αMHC-Cre-driven deletion caused perinatal lethality, they used AAV9-cTnT-Cre injected at postnatal day 1 to achieve mosaic deletion at varying efficiencies (high dose: ~80% of cardiomyocytes; medium: ~60%; low: ~40%). They assessed cardiac function by echocardiography, cardiomyocyte maturation by confocal imaging of T-tubules (using FM 4-64FX perfusion and the mTmG lineage reporter), and gene/protein expression by qPCR and western blot. Metabolic function was evaluated by measuring mitochondrial respiration (Seahorse), ATP content, lipid accumulation (Oil Red O, LipidTOX, BODIPY, triglyceride assays), and untargeted metabolomics. RNA-seq was performed on αMHC-Cre-driven NAE1 knockout hearts at embryonic/neonatal stages. HIF1α regulation was studied using immunoprecipitation, denaturing IP, and Cul2 siRNA knockdown.

**Key Results:** High-dose AAV-Cre-mediated NAE1 deletion caused progressive dilated cardiomyopathy and heart failure by 6 weeks of age, with increased heart weight-to-body weight ratio, enlarged LV chamber, reduced wall thickness, and declining ejection fraction and fractional shortening. No increase in cardiomyocyte apoptosis was detected. At lower AAV doses that did not cause overt cardiac dysfunction, NAE1-deficient cardiomyocytes showed severely disorganized T-tubules, reduced cell area, increased length-to-width ratio, and persistent expression of fetal gene isoforms (Myh7, Tnni1, Myl7, Hcn4) with downregulation of adult isoforms (Myh6, Tnni3, Myl2, Kcnj2). RNA-seq of NAE1-deficient hearts revealed enrichment of immature gene signatures and downregulation of oxidative phosphorylation and fatty acid metabolism genes, with upregulation of hypoxia and glycolysis pathways. Mitochondrial respiration was substantially reduced in NAE1-deficient hearts (basal and maximal respiration both decreased), and ATP content fell to nearly half of control levels. In cultured neonatal rat ventricular cardiomyocytes, NAE1 inhibition (by MLN4924 or siRNA) impaired fatty acid utilization, increased lipid accumulation, and suppressed OA-induced upregulation of Cpt1b, Atgl, Pgc1a, and Pgc1b. Metabolomics identified 413 significantly altered metabolites, with top enriched pathways including fatty acid biosynthesis, carnitine synthesis, Warburg effect, and glycolysis. Mechanistically, NAE1 deletion led to accumulation of HIF1α protein (without changes in its mRNA) and its nuclear localization in embryonic day 14.5 hearts. HIF1α target genes involved in fatty acid metabolism and mitochondrial function were downregulated. The authors demonstrated that neddylation regulates HIF1α through dual mechanisms: (1) indirectly via neddylation of Cul2 (a component of the VHL ubiquitin ligase complex that targets HIF1α for degradation), and (2) directly via neddylation of HIF1α itself, as shown by denaturing immunoprecipitation. MLN4924 remained effective in stabilizing HIF1α in Cul2-deficient cardiomyocytes, confirming a Cul2-independent mechanism. Pharmacological inhibition of HIF1α with echinomycin reduced MLN4924-induced lipid accumulation.

**Clinical Implications:** This study identifies neddylation as a critical post-translational mechanism driving cardiac metabolic maturation. The finding that neddylation promotes HIF1α degradation—both through Cul2-dependent and independent pathways—provides a mechanistic framework for understanding how the fetal-to-adult metabolic switch is controlled. These insights could inform strategies to improve the maturation of iPSC-derived cardiomyocytes for regenerative medicine, where inadequate metabolic maturation remains a major limitation. Additionally, the results suggest that neddylation pathway components could be therapeutic targets for congenital heart diseases linked to defective cardiac maturation. The study also highlights the potential cardiotoxicity of NAE1 inhibitors (e.g., MLN4924, which is in clinical trials for cancer) when used in pediatric populations during critical developmental windows.