Abstract Body: Introduction: Hypertension is a major risk factor for heart disease and stroke, and even when optimally treated, hypertensive patients still have a 50% greater cardiovascular risk than untreated normotensive subjects. Appropriate vascular smooth muscle cell (VSMC) cytoskeletal organization is essential for proper maintenance of tension, force generation, and vascular contractility and requires a precise stoichiometry of contractile and sarcomeric proteins. Post-transcriptional modification of mRNA allows the VSMC to fine-tune its response to external stimuli, and proteins that regulate mRNA stability are understudied, yet key targets in the regulation of vascular disease and especially contractility. The role of METTL14, a major m6A methylase, in VSMC contractile regulation is unknown. This work aims to test the hypothesis that METTL14 and m6A mRNA modification is a major mediator of Angiotensin II (AngII)-induced cytoskeletal dynamics and regulates VSMC contractile response.
Results: METTL14 protein abundance is significantly decreased in aorta of mice infused for two weeks with AngII compared with mice infused with saline. However, AngII stimulation increases abundance of m6A mRNA modification in hVSMCs. Using m6A MeRIP-sequencing, we quantified the degree of m6A methylation stoichiometry and identified hVSMC mRNA transcripts that are m6A methylated in response to AngII. Transcripts related to cytoskeletal-dependent processes are more highly m6A-modified compared to other mRNAs, and knockdown of METTL14 in AngII stimulated cells resulted in altered methylation status, mRNA abundance, and mRNA stability of mRNA related to various contractile processes. Knockdown of METTL14 in primary human aortic VSMCs dysregulates AngII-induced VSMC contraction in a collagen plug contraction assay as well as VSMC migration, which are both cytoskeletal-dependent processes.
Conclusion: Our data suggest that METTL14 may play a role in hypertension and regulation of vascular contractility by altering the abundance of cytoskeletal-related RNA and protein, thereby disrupting proper contractile mechanisms in VSMCs. This information will define a role of mRNA methylation, and METTL14 in particular, in regulation of VSMC contractile dynamics that may serve as a potential therapeutic target to regulate blood pressure and suggests that mRNA methylation of contractile-associated mRNAs in VSMCs may be an important, and potentially druggable process in development of hypertension.