Ventricular arrhythmias are thought to cause a majority of cases of sudden cardiac death, a significant contributor to deaths worldwide. A detailed understanding of the underlying mechanisms on a molecular and regulatory level is required to develop novel therapeutic approaches. Long noncoding RNAs (lncRNAs), a class of RNAs longer than 200 nucleotides with limited coding potential, have been reported to act as such regulatory elements. While a few lncRNAs are known to affect the cardiac rhythm, most have been characterized in a rodent model, limiting the applicability of these findings to patients.

To identify lncRNAs involved in regulating the human cardiac rhythm, we performed deep RNA-sequencing in human iPSC-derived cardiomyocytes (hiPSC-CMs). To supplement these data, we reanalyzed publicly available transcriptomic datasets of human adult left ventricular myocardium. LncRNAs exhibiting strong expression in both datasets were selected for further analysis. We ranked promising lncRNA candidates by evaluating their likely relevance to arrhythmia, either by considering their genomic locus and its relation to known protein-coding arrhythmia-genes or leveraging data from genome-wide association studies with relevance to ECG-abnormalities and rhythm disorders. In addition, cell type-specific expression, as determined using publicly available single-nuclear RNA-sequencing datasets and the likelihood of conservation in mammals were deciding parameters in our selection.

To determine whether manipulation of the top seven lncRNAs resulted in a phenotype in vitro, we utilized the RNA-targeting CRISPR-CasRx system. For this purpose, we designed vectors coding for both the enzyme and a sgRNA-array targeting each candidate with three specific sgRNAs. We delivered these into hiPSC-CMs using adeno-associated virus serotype 6 (AAV6). As a proof-of-concept experiment, we successfully utilized this strategy on the ion channel KCNQ1, examining the effect on both the RNA-level and on a phenotypic level.

For 6 out of 7 lncRNA candidates, we observed robust repression of the target lncRNA levels of 50-90 % using quantitative PCR. Using the voltage-sensitive dye di-8-ANEPPS, we performed optical action potential (AP) recordings of hiPSC-CMs, revealing significant AP morphology changes after manipulation of 5 lncRNA-candidates. For instance, the repression of one of the most promising candidates, TRDN-AS1, caused a significant prolongation of the AP duration by 50 ms (15 % increase).

In summary, we identified several lncRNA candidates that may be involved in regulating the human cardiac rhythm. In addition, we established a pipeline to screen these lncRNAs on a phenotypic level. Upon further validation, we will continue to characterize their mechanism of action and investigate their potential role as novel drug targets.