Abstract Body: Background: RNA localization in cardiomyocytes (CMs) has gained interest with recent evidence of local RNA enrichment and translation at sarcomeres and intercalated discs (ICDs), with microtubule-dependent mRNA localization appearing to be required for cardiac hypertrophy. To date, the identities of ICD-localized mRNAs has remained largely unknown, as no transcriptome-wide unbiased approaches have been used to spatially profile RNAs at ICDs; in addition, few efforts have been made to identify the specific RNA-binding proteins (RBPs) and/or trafficking machinery that mediate ICD RNA localization.
Methods: We developed an “ICD-seq” protocol to isolate and sequence RNAs enriched at ICDs using laser-capture microdissection from wildtype (WT) mouse heart tissue sections (n=11). This required troubleshooting to preserve RNA integrity during ICD immunostaining. ICDs were laser-captured with a 5 µm margin, and control regions were captured either from areas within individual CMs sparing ICDs or as large sectional areas of CM dense regions. RNA isolation and library prep kits designed for low inputs were used to generate RNA-seq libraries. Hybridization chain reaction fluorescence in situ hybridization (HCR-FISH) was performed to validate ICD-localization of “top-hit” mRNAs. Bioinformatic tools were used to identify enriched sequence motifs and candidate RBPs. We studied CM-specific APC knockout (cApc^-/-) mice by injecting AAV:Tnnt2-Cre virus into 4-week-old Apc^fl/fl BL6J mice and WT littermates (n=10 each) and assessed survival and cardiac function by echo- and electro-cardiogram.
Results: We identified ~800 ICD-enriched transcripts (FDR<0.05). Functional classification by pathway analysis yielded terms related to cardiac growth and structure as well as intriguing enrichment of transcriptional regulators. We found significant 4-fold enrichment of G/AG-rich motifs, consistent with high abundance of RNA G-quadruplex structures (rG4s) among ICD-enriched transcripts (p=2.1^e-30). We also noted that ICD RNAs were enriched for those known to be trafficked by APC/KIF1C pathway, and notably, cApc^-/- mice develop severe dilated cardiomyopathy and early death (43-day median survival post:AAV-Cre, p<0.0001).
Conclusion: We developed a working ICD-seq protocol and found specific pools of ICD-enriched transcripts in CMs, with a significant portion likely trafficked and localized by rG4- and APC/KIF1C-dependent mechanisms essential for normal cardiac structure and function.