RNA splicing controls organ-wide maturation of postnatal heart in mice

Postnatal cardiac development requires the orchestrated maturation of diverse cellular components, for which unifying control mechanisms are still lacking. Using full-length sequencing, we examined the transcriptomic landscape of the maturating mouse heart (E18.5-P28) at single-cell and transcript isoform resolution. We identified dynamically changing intercellular networks as a molecular basis of the maturing heart, and alternative splicing (AS) as a common mechanism that distinguished developmental age. Manipulation of RNA-binding proteins (RBPs) remodeled the AS landscape, cardiac cell maturation, and intercellular communication through direct binding of splice targets, which were enriched for functions related to general, as well as cell type-specific, maturation. Overexpression of an RBP NCBP2 in neonatal hearts repressed cardiac maturation. Together, our data suggest AS regulation by RBPs as an organ-level control mechanism in mammalian postnatal cardiac development, and provide insight into the possibility of manipulating RBPs for therapeutic purposes. Overall design: We designed a time course covering all critical postnatal (P) stages up until early adulthood (P1-7, P10, P14, and P28) ans isolate the cardiomyocytes and non-cardiomyocytes for single-cell sequencing. CLIP-seq data of G3BP1, NCBP2 and PTBP1 of cardiomyocytes, endothelial and fibroblast from nenatal heart. Different Lentivirus or adenovirus were concentrated, and the efficiencies were tested in P1 CMs, ECs, and FBs in vitro. For transduction, 200 ?l lentivirus and 8 µg/ml polybrene or 4 ?l adenovirus were added to cultured P1 CMs, ECs, FBs and adult CMs . Twenty-four hours later, cell medium was changed to DMEM supplemented with 10% FBS (Gibco, 11965092), 1% penicillin-streptomycin (Gibco, 15140122), and 5 µg/ml puromycin (Thermo, A1113802) for CMs and FBs or DMEM/F12 (Gibco, 11330032) supplemented with 10% FBS (Gibco, 11965092), 1% penicillin-streptomycin (Gibco, 15140122), 1 × ECGS (ScienceCell, 1052), and 5 µg/ml puromycin (Thermo, A1113802) for ECs. Forty-eight hours post transduction, cells were collected for subsequent experiments. AAV expressing GFP (control), Ncbp2, G3bp1, or Ptbp1 were directly injected into the anterior myocardial wall of P1 mice. Validation of overexpression by qPCR and immunofluorescence (IF) staining were performed at age P14 (2 weeks, 2W) and assessment of cardiac function by echocardiography was performed at 2W and P35 (5 weeks, 5W) for all RBPs, respectively. At endpoint (5W), scRNA-seq was performed for GFP OE and Ncbp2 OE hearts.