Sub-cellular cardiac in vivo murine proteomics, unravels the sarcomere and ribo-interactome with skNAC contribution to mitochondrial proteome

Adult cardiomyocytes are spatially organized cells with localized translational domains adjacent to the Z-disc of the sarcomere. However, the specific subcellular location of the proteins responsible for the folding, processing, and transport of newly translated proteins, as well as their role in protein sorting within cardiomyocytes, remains unknown.
I performed in vivo proximity dependent biotin labeling in adult mice, and delineated the subcellular proteome across different sarcomeric, ribosomal and intercalated disc nano-domains. Through this approach I identified the localization of sarcomeric and ribosomal associated proteins and validated the data by developing a universal in vivo CRISPR dependent approach termed Homology-independent targeted integration (HITI). Among them I identified the muscle specific isoform of the alpha subunit of nascent polypeptide associated complex (skNAC) as a Z-disc and ribosomal interactome. HITI mediated cardiomyocyte specific knock-out of skNAC, that preserved the expression of the short ubiquitous NACA isoform resulted in dilated cardiomyopathy phenotype with specific loss of nuclear encoded mitochondrial proteins. Affected cardiomyocytes demonstrated altered ribosomal positioning suggesting skNAC is responsible for the precise localization of the ribosomes and for the sorting of mitochondrial proteins.
My findings map the adult cardiomyocytes sub-cellular proteome and identify the role of skNAC isoform in ribosome localization, sorting of nuclear encoded mitochondrial proteins, and cardiac function.