Periodic Reporting for period 3 - CodingHeart (Novel Coding Factors in Heart Disease)
Período documentado: 2022-01-01 hasta 2023-06-30
Throughout this project, we want to discern fundamental causes of maladaptive responses in the heart as well as open strategies to monitor and limit them. To solve all these scientific questions we follow four main objectives: (i) we aim to detect short open reading frames (sORFs) in transcribed lncRNAs and circRNAs in human and rat hearts, (ii) we planned a computational and single cell sequencing based micropeptide candidate selection, (iii) we plan high-throughput in vitro and in vivo characterization of candidate micropeptides, and (iv) we finally aim to define disease mechanisms and pathophysiological consequences of our detected novel micropeptides.
To elucidate the biological role of detected micropeptides, we investigated several micropeptide features. These features include their expression pattern across tissues and cardiac cell types, secondary structure, differential expression in diseased hearts, and conservation. We found seventeen of the 169 translated lncRNAs to be specifically expressed in the heart based on GTEx expression data. Using the latest single cell technologies, we were able to generate a comprehensive single-nucleus and non-myocyte single-cell dataset, derived from 6 different atrial and ventricular locations of 14 different donor hearts (Litvinukova et al, Nature, 2020). We applied downstream statistical analysis on our single-cell atlas of the adult human heart to define cell-type specificity of identified translated lncRNAs. We found several lncRNAs to be cell-type specifically expressed, with the majority of cardiac-specific translated lncRNAs assigned to cardiomyocytes. In silico prediction of cleavage sites and secondary structures revealed 3 micropeptides that are potentially secreted, indicating a potential role as signaling molecules, and 7 micropeptides that might contain transmembrane domains, a feature often observed across the few known and biologically characterized micropeptides. Moreover, we find 34 and 7 micropeptides that are up- and downregulated in dilated cardioymyopathy (DCM) hearts, respectively. We only detect 17 micropeptides with strong sequence conservation to vertebrates based on PhyloCSF scores, but can align many lncRNAs to the genomes of other hominid species (chimp, gorilla, and orangutan; n = 79) or to the genomes of other primates or mammals (n = 31 or 43, respectively), with 16 being completely specific to humans (van Heesch et al., 2019). We found 46 micropeptides were localized to mitochondria, highlighting a potential involvement in cardiac energy metabolism. Additionally, we have designed and performed a high-throughput, peptide array based interactome screen termed PRISMA (protein interaction screen on peptide matrix). This screen is currently being analyzed, we have first results that are concordant with the detected localization of these micropeptides.