DSP-AS1 lncRNA as a promising target for treating desmoplakin cardiomyopathy

Abstract Background Arrhythmogenic cardiomyopathy (ACM) is an inherited cardiac disorder characterized by the progressive loss of cardiomyocytes, fibro-fatty replacement and ventricular arrhythmias (1). Desmoplakin cardiomyopathy (DSP-CM) has recently been recognised as a distinct form of ACM that more frequently affects the left ventricle or both ventricles (2). Pathogenic variants in the DSP gene, predispose to severe phenotypes with extensive fibrosis, high arrhythmic risk and recurrent myocardial injury. We previously showed that the DSP antisense lncRNA (DSP-AS1), which partially overlaps the DSP locus, reduces DSP transcript and protein levels in hiPSC-derived cardiomyocytes (iCMs). We also demonstrated that a Gapmer (LNA2) increases DSP transcripts by downregulating DSP-AS1 (3). Since several mutations in the DSP gene cause haploinsufficiency, targeting DSP-AS1 could be a potential therapy for DSP-CM. Purpose 1) To test the LNA2 effect on DSP iCMs 2) To develop advanced DSP-CM cardiac models for LNA2 testing. Methods hiPSCs from a DSP-CM patient, carrying a heterozygous nonsense mutation leading to DSP haploinsufficiency, and the isogenic control line (4) were differentiated in iCMs. The LNA2 GapmeR was used to downregulate the DSP-AS1 expression in DSP iCMs. After 10 days of treatment iCMs lysates were collected and analysed by ddPCR. DSP hiPSCs and the isogenic line were then differentiated in cardiac fibroblasts (iCFs) and endothelial cells (iECs) to create, together with iCMs, multicellular microtissues (MTs) and engineered heart tissues (EHTs). ddPCR was used to test the expression of DSP-AS1 and DSP in these cells. Results DSP-AS1 and DSP expression were confirmed by ddPCR in iCMs, iCFs and iECs, showing a clear condition of DSP haploinsufficiency. DSP-AS1 and DSP transcripts levels were higher in iCMs and iCFs, compared to iECs, suggesting that iECs are probably less involved in the DSP-AS1/DSP regulatory axis. Importantly, administration of LNA2 to DSP iCMs produced a significant decrease in DSP-AS1 expression, while concurrently restoring DSP transcript levels. In 2D multielectrode array (MEA) recordings, DSP iCMs exhibited a prolonged field potential duration (FPDc) compared with its isogenic control. Moreover, the DSP iCMs showed a blunted response to β-adrenergic stimulation with isoproterenol, displaying a markedly reduced FPD shortening. Finally, we successfully generated the first multicellular EHTs and MTs. Of note, DSP EHTs showed arrhythmic events, underlined by a significant contractile variability. Conclusions These preliminary findings suggest that: 1) DSP-AS1 inhibition by LNA2 can effectively restore DSP transcript levels in DSP iCMs 2) Multicellular EHTs recapitulate the arrhythmic phenotype typical of the disease. Further molecular and electrophysiological studies are needed to validate the efficacy of LNA2 approach first in iCMs and then in the 3D multicellular cardiac models.