Fibro-adipose substitution has a double detrimental effect on the myocardium in arrhythmogenic cardiomyopathy (ACM), worsening arrhythmogenesis by creating a non-conductive substrate, and causing ventricular dysfunction leading to heart failure. Notably, to-date no etiological therapy is available. This work introduces, for the first time, the stromal cardiac compartment as a key player in ACM ventricular adipose substitution: we demonstrated that cardiac human mesenchymal stromal cells undergo adipogenic differentiation both in ACM explanted hearts and in culture through a PKP2-dependent mechanism. Cardiac mesenchymal stromal cells constitute a suitable cellular platform for future mechanistic studies and a potential target for future therapies.

Rationale: microRNAs (miRNAs) modulate gene expression by repressing translation of targeted genes. Previous work has established a role for miRNAs in regulating vascular smooth muscle cell (VSMC) activity. Whether circular RNAs are involved in the modulation of miRNA activity in VSMCs is unknown. Objective: We aimed to identify circular RNAs interacting with miRNAs enriched in VSMCs and modulating the cells’ activity. Methods and Results: RNA sequencing and bioinformatics identified several circular RNAs enriched in VSMCs; however, only one, possessing multiple putative binding sites for miR-145, was highly conserved between mouse and man. This circular RNA gemmed from alternative splicing of Lrp6 (lipoprotein receptor 6), a gene highly expressed in vessels and implicated in vascular pathologies and was thus named circ_Lrp6. Its role as a miR-145 sponge was confirmed by determining reciprocal interaction through RNA immunoprecipitation, stimulated emission depletion microscopy, and competitive luciferase assays; functional inhibition of miR-145 was assessed by measuring expression of the target genes ITGβ8 (integrin-β8), FASCIN (fascin actin-bundling protein 1), KLF4 (Kruppel-like factor 4), Yes1 (YES proto-oncogene 1), and Lox (lysyl oxidase). The interaction was preferentially localized to P-bodies, sites of mRNA degradation. Using loss- and gain-of-function approaches, we found that circ_Lrp6 hindered miR-145-mediated regulation of VSMC migration, proliferation, and differentiation. Differential expression of miR-145 and circ_Lrp6 in murine and human vascular diseases suggests that the ratio of circ_Lrp6 bound to miR-145 versus unbound could play a role in vascular pathogenesis. Viral delivery of circ_Lrp6 shRNA prevented intimal hyperplasia in mouse carotids. Conclusions: circ_Lrp6 is an intracellular modulator and a natural sponge for miR-145, counterbalancing the functions of the miRNA in VSMCs.

Rationale: MicroRNAs (miRNAs, miRs) are small noncoding RNAs that modulate gene expression by negatively regulating translation of target genes. Although the role of several miRNAs in vascular smooth muscle cells (VSMCs) has been extensively characterized, the function of miRNA-128-3p (miR-128) is still unknown. Objective: To determine if miR-128 modulates VSMC phenotype and to define the underlying mechanisms. Methods and Results: We screened for miRNAs whose expression is modulated by an altered DNA methylation status in VSMCs, and among the hits, we selected miR-128. We found that miR-128 was expressed in various tissues, primary murine cells, and pathological murine and human vascular specimens. Through gain- and loss-of-function approaches, we determined that miR-128 affects VSMC proliferation, migration, differentiation, and contractility. The alterations of those properties were dependent upon epigenetic regulation of key VSMC differentiation genes; notably, Kruppel-like factor 4 was found to be a direct target of miR-128 and able to modulate the methylation status of the pivotal VSMC gene myosin heavy chain 11 ( Myh11 ). Finally, in vivo lentiviral delivery of miR-128 prevented intimal hyperplasia in a mouse model of carotid restenosis without modifying vital cardiovascular parameters. Conclusion: miR-128 is a critical modulator of VSMCs and is regulated by epigenetic modifications upon stress. Its modulation in the context of disease could be exploited for therapeutic purposes.

No abstract