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Abstract

Recent studies of cardiac translatomes have revealed widespread translation of small open reading frames into microproteins. Once described as translational noise, these peptides are now recognized as intriguing new players in diverse biological processes. Motivated by their prevalence, I adopted a ribosome profiling-based approach and applied it to mouse myocardium and human heart biopsies to search for previously uncharacterized cardiac microproteins. In addition, heart translatomes were compared with proteomics data and combined with conservation analysis. Integration of these results revealed that novel cardiac microproteins are rare occurrences with little sign of conservation across mammals. Instead, focusing on annotated but poorly characterized small proteins in the heart led to the identification of cardiac-enriched microprotein termed small integral membrane protein 4 (SMIM4). This promising candidate was used for initial functional characterization, which demonstrated the role of SMIM4 in cardiomyocyte viability, mitochondrial metabolism and growth. Further work will be conducted in vivo, to understand the contribution of this mitochondrial peptide to heart physiology and cardiac response to stress. Another recently identified microprotein is small regulatory polypeptide of amino acid response (SPAAR), which features particularly high abundance in murine and human hearts. Its restricted expression pattern and inhibitory effect on mechanistic target of rapamycin complex 1 (mTORC1) suggest that SPAAR plays an important role in regulating cardiac function. Therefore, I studied the role of SPAAR in the heart by using a CRISPR/Cas9-generated knockout mouse model (SPAAR KO), which carries a deletion of the start codon to prevent SPAAR translation, but maintains constant transcript levels. Inducing myocardial ischemia-reperfusion injury in these animals revealed that SPAAR KO mice are protected from myocardial damage early on, as they demonstrated decreased levels of biomarkers for cardiac injury. This protective effect continued at later time points and resulted in sustained left ventricular ejection fraction in SPAAR KO mice, accompanied by reduced infarct sizes. Strikingly, published single-cell RNA-sequencing data of mammalian hearts indicate enrichment of Spaar in cardiac endothelial cells. To understand the molecular mechanisms of SPAAR function, I then focused on the characterization of cardiac endothelial cells. Taking various approaches, I identified several potential targets for mediating SPAAR function. These cell type-specific observations suggest the modulation of cardiac endothelial function by microprotein SPAAR. Combined with the cardioprotective effect after ischemia-reperfusion injury in vivo, these findings could prove clinically relevant in the context of ischemic cardiac disease.