Abstract Body: Heart failure with preserved ejection fraction (HFpEF) accounts for more than half of all HF, yet effective therapies remain limited. Metabolomics of myocardial biopsies from patients with HFpEF revealed spermidine the most upregulated. Spermidine is critical to ribosomal translation due to its engagement with hypusination of the post-translation modification of eukaryotic translation initiation factor 5A (eIF5A^Hyp). We find eIF5A^Hyp to be decreased in HFpEF but not HFrEF, associated with reduced expression of the primary synthetic enzyme deoxy hypusine synthase (DHPS). Accordingly, mice with cardiomyocyte specific conditional knockout of DHPS (cDHPS-KO) were generated and display preserved ejection fraction yet increased E/E’, pulmonary edema, and exercise intolerance. Ribosome profiling and sequencing was performed to assess transcripts with impaired translation, and among those identified, gene ontology highlighted ubiquitin related pathways. CRISPR knock-out of DHPS in neonatal rat cardiomyocytes reduces eIF5A^Hyp, total ubiquitinated proteins, and free ubiquitin. Reducing eIF5A^Hyp pharmacologically and genetically leads to accumulation of protein aggregates in cardiomyocytes but they are less ubiquitinated. The ubiquitin precursor, UBC encoding 9 ubiquitin monomers, and deubiquitinases, USP7, USP16, and USP9X, are required to generate free ubiquitin, and all have reduced expression in myocytes with reduced eIF5A^Hyp, in cDHPS-KO hearts, and human HFpEF myocardium. Transfection of recombinant single ubiquitin suppresses toxicity seen in cardiomyocytes with reduced eIF5A^Hyp. In conclusion, we have identified impaired protein quality control that is unique to HFpEF, caused by reduced eIF5A^Hyp that in turn impairs translation of UBC and key deubiquitinases required to generate free ubiquitin.