Abstract 4369500: MicroRNA-1’s Biophysical Function is essential for Postnatal Cardiac Maturation

Abstract

Background: MicroRNA-1 (miR1) plays a critical role in maintaining cardiac homeostasis. Full knockout of miR1 results in postnatal lethality. While prior studies have primarily focused on the canonical RNAi mechanism, we recently discovered a novel biophysical function of miR1, whereby it modulates the activity of its-bound proteins. Notably, a human single nucleotide polymorphism 14A/G of miR1 selectively disrupts the biophysical action without affecting its RNAi, indicating their mechanistic independence. Objective: To investigate the specific physiological significance of miR1’s biophysical function in cardiac development and function. Methods: Using CRISPR/Cas9 genome editing, we introduced a 14A/G point mutation into both miR1 gene loci and successfully generated homozygous transgenic mice (14G-Homo, miR1-1 14G/G ; miR1-2 14G/G ). Cardiac function was assessed longitudinally by echocardiography from postnatal-day 0.5 (P0.5) to 8 weeks. Neonatal cardiomyocytes (CMs, P1.5) were isolated for functional analyses. Results: Wild-type (WT) mice demonstrated normal postnatal cardiac growth, with left ventricle (LV) ejection fraction (EF) remaining stable at ~70% during the first postnatal week, followed by a decline to ~ 60% by P11.5. Stroke volume (SV) increased progressively, with a notable acceleration beginning at P11.5. In contrast, 14G-Homo mice exhibited significantly reduced EF as early as P0.5 (52±1.96% vs. WT 71±2.20%, p = 2.2×10 -5 ), despite a transient normalization by P3.5 (67±2.08% vs. WT 71±1.72%, p = 0.15). The accelerated increase in SV was markedly delayed in 14G-Homo mice until P14.5, resulting in significantly decreased SV (4.44±0.644��l vs. WT 10.68±0.401µl, p =1.6×10 -7 ). Pulse wave Doppler analysis showed that the E/A ratio reversal (≥1), a hallmark of enhanced LV diastolic function, occurred at P4.5 in WT mice. This reversal was delayed until P8.5 in 14G-Homo mice, indicating a delay in cardiac functional maturation. In vitro, WT neonatal CMs showed significant hypertrophic response to phenylephrine (PE), whereas both 14G-Homo CMs and WT CMs transfected with mutated 14G-miR did not. Importantly, reintroduction of WT miR1 restored PE-induced hypertrophy in 14G-Homo CMs. Conclusion: The biophysical function of miR1 is essential for timely postnatal cardiac maturation. These findings reveal a previously unrecognized mechanism of miRs and suggest that targeting its biophysical interactions may provide new therapeutic strategies for heart disease.