Background
N
6
-methyladenosine (m
6
A) 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 m
6
A-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 m
6
A 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 m
6
A demethylation in the 3’UTR of
Notch1
mRNA and elevated m
6
A mRNA levels. These elevated m
6
A levels in
Notch1
mRNA markedly enhanced YTHDF2 phase separation, increased the recognition of m
6
A 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 m
6
A-mediated phase separation sup...
