AIMS: Circular RNA (circRNA) is a newly validated class of single-stranded RNA, 
ubiquitously expressed in mammalian tissues and possessing key functions 
including acting as microRNA sponges and as transcriptional regulators by 
binding to RNA-binding proteins. While independent studies confirm the 
expression of circRNA in various tissue types, genome-wide circRNA expression in 
the heart has yet to be described in detail.
METHODS AND RESULTS: We performed deep RNA-sequencing on ribosomal-depleted RNA 
isolated from 12 human hearts, 25 mouse hearts and across a 28-day 
differentiation time-course of human embryonic stem cell-derived cardiomyocytes. 
Using purpose-designed bioinformatics tools, we uncovered a total of 15 318 and 
3017 cardiac circRNA within human and mouse, respectively. Their abundance 
generally correlates with the abundance of their cognate linear RNA, but 
selected circRNAs exist at disproportionately higher abundance. Top highly 
expressed circRNA corresponded to key cardiac genes including Titin (TTN), RYR2, 
and DMD. The most abundant cardiac-expressed circRNA is a cytoplasmic localized 
single-exon circSLC8A1-1. The longest human transcript TTN alone generates up to 
415 different exonic circRNA isoforms, the majority (83%) of which originates 
from the I-band domain. Finally, we confirmed the expression of selected cardiac 
circRNA by RT-PCR, Sanger sequencing and single molecule RNA-fluorescence in 
situ hybridization.
CONCLUSIONS: Our data provide a detailed circRNA expression landscape in hearts. 
There is a high-abundance of specific cardiac-expressed circRNA. These findings 
open up a new avenue for future investigation into this emerging class of RNA.