Intercalated discs (ICDs) are dynamic and complex structures that couple cardiomyocytes (CMs) electrically and mechanically to synchronize contraction, which is vital for heart pump function. Maintenance of ICD structure and protein localization at ICDs is essential for CM function, and dysregulation of ICD structure and protein composition is implicated in heart disease [e.g. dilated cardiomyopathy, heart failure (HF), and arrhythmias]. Beyond their known structural roles, ICDs also appear to serve as hubs for local protein synthesis. Studies have found enhanced local translation at ICDs, but only a few select ICD-enriched mRNAs are known (e.g. Dsp; encodes an essential ICD protein). Overall, mRNA transport and local translation have been shown to be biologically important in many cells and organisms, and dysregulation of these processes are detrimental to cell function. Despite evidence for both ICD localized RNAs and local translation in CMs, no transcriptome-wide unbiased approaches have been used to profile RNAs at ICDs, and few efforts have been made to identify the RNA-binding proteins (RBPs) and/or trafficking machinery that mediate RNA localization to ICDs. To begin addressing this, we developed “ICD-seq”, overcoming several technical and methodological hurdles to enable high-throughput laser capture microdissection of immunostained ICDs from fresh-frozen cardiac tissue sections. We successfully captured >20,000 ICDs from mouse heart tissues, from which we subsequently isolated RNAs and performed RNA-seq. We identified hundreds of unique mRNAs that are significantly enriched (and de-enriched) at ICDs (Fig. 1) and found that enriched mRNAs harbor sequence motifs known to bind several specific RBPs, some of which are implicated in heart disease. Gene ontology analyses indicate that many ICD-enriched mRNAs encode proteins involved in RNA metabolism, microtubule stability and transport, and cell-cell adhesion. Furthermore, ICD-localized mRNAs are primarily enriched for mRNAs that are known to be trafficked to cell leading edges in APC/Kif1c-dependent fashion (see Resubmission Modifications, Fig. S1). Notably, initial pilot studies applying ICD-seq to nonfailing human heart tissues revealed a strong correlation to mouse data supporting conservation and functional importance. Based on these overall initial observations, our overarching hypothesis is that select subsets of functionally related mRNAs are trafficked to, anchored, and translated at ICDs – via conserved mechanisms that depend on microtubules and an array of molecular motors and RBPs and that these processes are perturbed during cardiac stress and contribute to detriments in heart function.

Honors/Awards:

Graduate College Summer Fellowship (2024)