N6-Methyladenosine-mediated phase separation suppresses NOTCH1 expression and promotes mitochondrial fission in diabetic cardiac fibrosis.

Background: N⁶-methyladenosine (m⁶A) modification of messenger RNA (mRNA) is crucial for liquid-liquid phase separation in mammals. Increasing evidence indicates that liquid-liquid phase separation in proteins and RNAs affects diabetic cardiomyopathy. However, the molecular mechanism by which m⁶A-mediated phase separation regulates diabetic cardiac fibrosis remains elusive.

Methods: Leptin receptor-deficient mice (db/db), cardiac fibroblast-specific Notch1 conditional knockout (POSTN-Cre × Notch1^flox/flox) mice, and Cre mice were used to induce diabetic cardiac fibrosis. Adeno-associated virus 9 carrying cardiac fibroblast-specific periostin (Postn) promoter-driven small hairpin RNA targeting Alkbh5, Ythdf2, or Notch1, and the phase separation inhibitor 1,6-hexanediol were administered to investigate their roles in diabetic cardiac fibrosis. Histological and biochemical analyses were performed to determine how Alkbh5 and Ythdf2 regulate Notch1 expression in diabetic cardiac fibrosis. NOTCH1 was reconstituted in ALKBH5- and YTHDF2-deficient cardiac fibroblasts and mouse hearts to study its effects on mitochondrial fission and diabetic cardiac fibrosis. Heart tissue samples from patients with diabetic cardiomyopathy were used to validate our findings.

Results: In mice with diabetic cardiac fibrosis, decreased Notch1 expression was accompanied by high m⁶A mRNA levels and mitochondrial fission. Fibroblast-specific deletion of Notch1 enhanced mitochondrial fission and cardiac fibroblast proliferation and induced diabetic cardiac fibrosis in mice. Notch1 downregulation was associated with Alkbh5-mediated m⁶A demethylation in the 3'UTR of Notch1 mRNA and elevated m⁶A mRNA levels. These elevated m⁶A levels in Notch1 mRNA markedly enhanced YTHDF2 phase separation, increased the recognition of m⁶A residues in Notch1 mRNA by YTHDF2, and induced Notch1 degradation. Conversely, epitranscriptomic downregulation rescues Notch1 expression, resulting in the opposite effects. Human heart tissues from patients with diabetic cardiomyopathy were used to validate the findings in mice with diabetic cardiac fibrosis.

Conclusions: We identified a novel epitranscriptomic mechanism by which m⁶A-mediated phase separation suppresses Notch1 expression, thereby promoting mitochondrial fission in diabetic cardiac fibrosis. Our findings provide new insights for the development of novel treatment approaches for patients with diabetic cardiac fibrosis.