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Exploration of adult mouse Single Cardiomyocyte Proteome using Label-Free LC-MS

ABSTRACT: Cardiac myocyte heterogeneity has been acknowledged based on ion channel expression, cellular electrophysiology and through transcriptomics, but the exploration of individual cardiomyocytes at the board proteome-wide level has remained challenging. We developed an entire analytical pipeline to assess the cell heterogeneity of adult primary cardiomyocytes isolated from mouse hearts. Over 700 single cell proteomes were isolated from 4 mice hearts. Each single cell image was captured by the cellenONE sorter interface and evaluated for morphological features of apoptotic cells, i.e.: hypercontracted cell shape. This imaging analysis allowed us to retain 270 rod-shaped cardiac cells for downstream single cell proteome sample processing and analysis on Bruker timsTOF SCP mass spectrometer. Final dataset UMAP dimension reduction and clustering analysis yielded 3 cardiomyocyte cell sub-populations that displayed differential: i) expression of Myh7 and Myh7b, ii) carbon metabolism signatures and iii) distribution of 7 proteins involved in calcium signaling. Single cardiomyocyte protemes demonstrated potential differences related to the metabolism and calcium ion handling between those sub-populations and could bring new insights for cardiology studies on mice.

INSTRUMENT(S): timsTOF SCP

ORGANISM(S): Mus Musculus (ncbitaxon:10090)

SUBMITTER: Jennifer Van Eyk

PROVIDER: MSV000093672 | MassIVE | Fri Dec 15 11:40:00 GMT 2023

SECONDARY ACCESSION(S): PXD047860

REPOSITORIES: MassIVE

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Project description:Background: Modulation of mRNA splicing acts as an important layer of gene regulation, in addition to transcriptional regulation and epigenetic modifications. RNA binding proteins (RBPs) play essential roles in mediating RNA splicing and are key regulators of heart development and function. Our previous studies demonstrated that RBPMS (RNA-binding protein with multiple splicing) regulates cardiac development through modulating mRNA splicing during embryogenesis. Here we explored the postnatal function of RBPMS in the heart. Methods: We ablated Rbpms in the heart by generating a cardiac-specific knockout mouse line (Myh6-Cre, Rbpmsfl/fl), and evaluated its cardiac functions by histology, echocardiography, and gene expression. Paired-end RNA sequencing and RT-PCR were performed to identify and validate splicing targets of RBPMS in adult mouse hearts. Proximity-dependent Biotin Identification (BioID) assay and mass spectrometry analysis were performed to identify RBPMS binding partners. We also measured contractility and calcium fluxes in isolated mouse cardiomyocytes, and contractile forces of cardiac papillary muscle. Human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs) were also used as a model to explore the influence of RBPMS on contractility of human cardiomyocytes. Results: he absence of Rbpms in the heart led to dilated cardiomyopathy (DCM) and heart failure, causing early death in mice. Mice with cardiac-specific knockout of Rbpms showed myocardium noncompaction with reduced cardiomyocyte number at the neonatal stage and developed DCM with pervasive myocardial fibrosis in adulthood. We found that RBPMS mediates a largely distinct RNA splicing profile in adult mouse hearts compared to neonatal hearts, indicating a stage-specific modulation of alternative RNA splicing by RBPMS. In adult hearts, RBPMS mainly influenced alternative splicing of genes associated with sarcomere structures and cardiomyocyte contraction, such as Ttn, Pdlim5 and Nexn, to generate new protein isoforms. In neonatal hearts, RBPMS influenced the splicing of cytoskeletal genes. RBMPS was associated with spliceosome factors and other RNA binding proteins that play important roles in the heart, such as RBM20 and GATA4. Importantly, we found that the absence of Rbpms caused severe cardiomyocyte contractile defects and reduced calcium sensitivity in both mouse and hiPSC-CMs. Our results demonstrated that Rbpms is crucial for postnatal cardiac function and cardiomyocyte contractility by regulating RNA alternative splicing. Conclusions: Loss of Rbpms in the heart causes reduced cardiomyocyte number and impaired cardiomyocyte contraction, leading to DCM and heart failure.

Project description:Cardiomyocytes exhibit differential growth patterns throughout development. In fetal life the increase in cardiac mass is associated with hyperplastic growth and cardiomyocyte proliferation. The majority of fetal cardiomyocytes are also mononucleated. During the early neonatal period in mice there is a switch from hyperplastic to hypertrophic growth of cardiomyocytes. This period is characterized by bi-nucleation and polyploidization of cardiomyocyte nuclei and a decreased capacity for cardiomyocytes to proliferate and complete cytokinesis. Increases in myocardial mass occur predominantly via hypertrophic growth. Adult mammalian cardiomyocytes are generally accepted to have little or no proliferative capacity and to be terminally withdrawn from the cell cycle. The vast majority of adult murine cardiomyocytes are bi-nucleated. The present study sought to accurately establish the growth pattern of cardiomyocytes throughout development in mice and identify genes associated with the switch from hyperplastic to hypertrophic growth. These cell cycle associated genes are crucial to the understanding of the mechanisms of bi-nucleation, polyploidization and hypertrophy in the neonatal period. Cardiomyocytes were FACS sorted from the hearts of ED11-12 embryos, neonatal day 3-4 and adult (10 week) eGFP ?-MHC mice whereby GFP expression is driven constitutively by the ?-MHC promoter. Gene analyses identified genes whose expression was predicted to be particular to day 3 -4 neonatal cardiomyocytes, compared to embryonic or adult cells. Cell cycle associated genes are crucial to the understanding of the mechanisms of bi-nucleation and hypertrophy in the neonatal period, and offer attractive candidates for manipulation. Total RNA obtained from isolated cardiomyocytes from ED11-12; Neonatal day 3-4 and adult timepoints compared with each other. Several hearts per sample, RNA was pooled within samples. FACS samples were prepared in the following manner: embryonic, neonatal and adult hearts were dissected and dissociated to single cell solution with Liberase Blendzyme 3 (0.1 mg/ml) (Roche Diagnostics), washed and spun down and resuspended in cardiomyocyte isolation buffer (130 mM NaCl; 5 mM KCl; 1.2 mM KH2PO4; 6 mM HEPES; 5 mM NaHCO3; 1 mM MgCl2; 5 mM Glucose).

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Exploration of adult mouse Single Cardiomyocyte Proteome using Label-Free LC-MS

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