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.
Aims We and others have previously described the expression landscape of circular RNA (circRNA) in mouse and human hearts. However, the functional relevance of many of these abundantly expressed cardiomyocyte circRNA remains to be fully explored. Among the most abundant circRNA, one stems from the sodium-calcium exchanger gene, Slc8a1, exon 2 locus. Because of its very high abundance in cardiomyocytes we investigated the possible role of circSlc8a1 in the heart. Methods and results We performed a miRNA screen using an array of 752 miRNAs with RNA recovered from a pull-down of endogenous cardiomyocyte circSlc8a1. MicroRNA-133a (miR-133a), with a prior well-recognized role in cardiac hypertrophy, was highly enriched in the fraction of circSlc8a1 pull-down (adjusted P-value < 0.001). We, therefore, followed-up validation of the functional interaction between circSlc8a1 and miR-133 using luciferase assays and reciprocal pull-down assays. In vivo, AAV9-mediated RNAi knockdown of circSlc8a1 attenuates cardiac hypertrophy from pressure-overload, whereas forced cardiomyocyte specific overexpression of circSlc8a1 resulted in heart failure. Molecular analyses showed targets of miR-133a including serum response factor (Srf), connective tissue growth factor (Ctgf), adrenoceptor beta 1 (Adrb1), and adenylate cyclase 6 (Adcy6) to be regulated by circSlc8a1-directed intervention of knockdown and overexpression. Conclusion In summary, circSlc8a1 can function as an endogenous sponge for miR-133a in cardiomyocytes. We propose that circSlc8a1 may serve as a novel therapeutic target for cardiac hypertrophy.
Circular RNAs (circRNAs) sequester microRNAs (miRNAs) and repress their endogenous activity. We hypothesized that artificial circRNA sponges (circmiRs) can be constructed to target miRNAs therapeutically, with a low dosage requirement and extended half-lives compared to current alternatives. This could present a new treatment approach for critical global pathologies, including cardiovascular disease. Here, we constructed a circmiR sponge to target known cardiac pro-hypertrophic miR-132 and -212. Expressed circmiRs competitively inhibited miR-132 and -212 activity in luciferase rescue assays and showed greater stability than linear sponges. A design containing 12 bulged binding sites with 12 nucleotides spacing was determined to be optimal. Adeno-associated viruses (AAVs) were used to deliver circmiRs to cardiomyocytes in vivo in a transverse aortic constriction (TAC) mouse model of cardiac disease. Hypertrophic disease characteristics were attenuated, and cardiac function was preserved in treated mice, demonstrating the potential of circmiRs as novel therapeutic tools. Subsequently, group I permutated intron-exon sequences were used to directly synthesize exogenous circmiRs, which showed greater in vitro efficacy than the current gold standard antagomiRs in inhibiting miRNA function. Engineered circRNAs thus offer exciting potential as future therapeutics.
Cardiac regeneration may revolutionize treatment for heart failure but endogenous progenitor-derived cardiomyocytes in the adult mammalian heart are few and pre-existing adult cardiomyocytes divide only at very low rates. Although candidate genes that control cardiomyocyte cell cycle re-entry have been implicated, expression heterogeneity in the cardiomyocyte stress-response has never been explored. Here, we show by single nuclear RNA-sequencing of cardiomyocytes from both mouse and human failing, and non-failing adult hearts that sub-populations of cardiomyocytes upregulate cell cycle activators and inhibitors consequent to the stress-response in vivo. We characterize these subgroups by weighted gene co-expression network analysis and discover long intergenic non-coding RNAs (lincRNA) as key nodal regulators. KD of nodal lincRNAs affects expression levels of genes related to dedifferentiation and cell cycle, within the same gene regulatory network. Our study reveals that sub-populations of adult cardiomyocytes may have a unique endogenous potential for cardiac regeneration in vivo.
Mutations in the LaminA gene are a common cause of monogenic dilated cardiomyopathy. Here we show that mice with a cardiomyocyte-specific Lmna deletion develop cardiac failure and die within 3–4 weeks after inducing the mutation. When the same Lmna mutations are induced in mice genetically deficient in the LINC complex protein SUN1, life is extended to more than one year. Disruption of SUN1’s function is also accomplished by transducing and expressing a dominant-negative SUN1 miniprotein in Lmna deficient cardiomyocytes, using the cardiotrophic Adeno Associated Viral Vector 9. The SUN1 miniprotein disrupts binding between the endogenous LINC complex SUN and KASH domains, displacing the cardiomyocyte KASH complexes from the nuclear periphery, resulting in at least a fivefold extension in lifespan. Cardiomyocyte-specific expression of the SUN1 miniprotein prevents cardiomyopathy progression, potentially avoiding the necessity of developing a specific therapeutic tailored to treating each different LMNA cardiomyopathy-inducing mutation of which there are more than 450.
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