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

Cardiovascular Diabetology volume 23, Article number: 347 (2024) Cite this article

Abstract

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 × Notch1flox/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.

Introduction

Diabetic cardiomyopathy (DCM) is a serious complication of diabetes that refers to a series of abnormalities in cardiac fibrosis and function [1]. Because cardiac fibroblast (CF) proliferation participates in extracellular matrix deposition, matrix maintenance, and cardiac fibrosis, it is important to determine the heterogeneous roles of fibroblasts in diabetic cardiac fibrosis [2]. Despite its poor prognosis [3], diabetic cardiac fibrosis still lacks formal treatment guidelines or approved drugs [4, 5]. Therefore, an in-depth understanding of the molecular mechanisms underlying CF proliferation in diabetic cardiac fibrosis is urgently needed.

Cellular proliferation is fueled by mitochondrial metabolic reprogramming, of which mitochondrial fission is a vital component [6]. Mitochondrial fission alters metabolism and proliferation [7] and is highly regulated by dynamin-related protein 1 (DRP1), mitochondrial fission factor (MFF), mitochondrial division (MID)49, MID51, and mitochondrial fission 1 (FIS1) proteins [8]. Abnormal mitochondrial fission is linked to neurodegeneration, cancer, pulmonary fibrosis, and diabetes mellitus [9]. However, the roles of mitochondrial fission in diabetic cardiac fibrosis and CF proliferation remain poorly understood.

Aberrant signaling of neurogenic locus notch homolog protein (NOTCH) and fibroblast proliferation [10] are associated with mitochondrial dysfunction [11]. The NOTCH1 signaling pathway regulates cardiac development and cardiomyocyte proliferation and differentiation [12]. The NOTCH1 intracytoplasmic domain (NICD1) translocates into the nucleus and regulates HES1 expression by binding to ubiquitous transcription factors and centromere-binding protein 1 [13]. The NOTCH1 signaling pathway also plays an important role in the genesis, development, and pathophysiology of the cardiovascular system [14]. However, whether NOTCH1 regulates mitochondrial fission, thereby contributing to CF proliferation in diabetic cardiac fibrosis, remains to be determined.

Dysregulated NOTCH1 modifications have been linked to epigenetic abnormalities [15]. N6-methyladenosine (m6A), the most abundant modification of mammalian RNAs, modulates almost all aspects of messenger RNA (mRNA) metabolism, including methylation, demethylation, and recognition [16]. Posttranscriptional modifications involve multiple proteins, including methyltransferase 3, N6-adenosine-methyltransferase complex catalytic subunit (METTL3), METTL14, WT1 associating protein (WTAP), KIAA1429, alkB homolog 5 RNA demethylase (ALKBH5), N6-methyladenosine RNA binding protein F (YTHDF)1/2/3, and YTHDC1/2 [17]. The m6A reader proteins YTHDF1, YTHDF2, and YTHDF3 contain putative prion-like domains and undergo liquid-liquid phase separation [18]. Liquid-like condensates are organized by multivalent, intrinsically disordered proteins and RNA molecules [19]. Interestingly, m6A enhances the phase separation potential of mRNAs [20]. Moreover, m6A methylation controls fibroblast proliferation and migration [21]. However, the molecular mechanisms of m6A-mediated phase separation that regulate diabetic cardiac fibrosis, fibroblast proliferation, and migration remain elusive.

Here, we addressed these fundamental questions by showing that ALKBH5 deficiency promotes mitochondrial fission and CF proliferation by increasing NOTCH1 methylation via YTHDF2-mediated modulation of m6A phase separation.

Methods

The detailed materials and methods are described in the Supplemental Material. All animal experiments followed protocols approved by the Ethics Committee of Anhui Medical University and the Second Affiliated Hospital of Anhui Medical University.

Animal models

All animal experimental protocols were conducted in accordance with the National Institutes of Health ‘Guidelines for the Use of Laboratory Animals’ and received approval from the Ethics Committee of Anhui Medical University and the Second Affiliated Hospital of Anhui Medical University.

In our study, we utilized the Leptin receptor-deficient (BKS-db/db) model, high fat diet (HFD)/streptozotocin (STZ) model, Notch1 conditional knockout (POSTN-Cre × Notch1flox/flox, Notch1-cKO)-HFD/STZ model, Notch cKO-AAV9 model, and db/db-AAV9 model.

Human samples

Samples were collected from 30 DCM patients and 30 non-diabetic healthy controls at the Second Affiliated Hospital of Anhui Medical University. This study was approved by the Ethics Committee of Anhui Medical University and the Second Affiliated Hospital. Inclusion criteria for DCM patients included diagnosed diabetes mellitus, absence of other causes of decreased left ventricular function, no history of hypertension, myocardial infarction, or suspected coronary heart disease, normal liver and kidney function, and normal lipids. The control group met inclusion criteria of normal blood lipid, blood pressure, and blood sugar levels, normal cardiac function and pulmonary artery pressure, and age and sex matching with the DCM group. After obtaining informed consent for surgery and follow-up, heart tissue was collected during heart valve replacement or cardiac catheterization and stored at -80 °C for further experiments.

Study approval

All animal procedures and experiments complied with NIH guidelines or Directive 2010/63/EU of the European Parliament on the Protection of Animals Used for Scientific Purposes and were approved by the ethics Committee of Anhui Medical University (The approval number is LLSC20232157). The clinical study for which written informed consent was obtained from all eligible patients, adhered to the principles of the Declaration of Helsinki; all experimental procedures involving human samples were reviewed and approved by the ethics committee of Anhui Medical University (The approval number is 2021061).

Statistical analysis

Statistical analyses were performed using GraphPad Prism v9.0 software. n represents the number of biological replicates. After the Kolmogorov-Smirnov Test confirmed data normality, differences between two groups were assessed using Student’s t-test, whereas multiple comparisons were performed using ANOVA followed by Bonferroni post hoc Tukey tests. All data are presented as mean ± standard error of the mean. Statistical significance was defined as P < 0.05.

Results

Decreased NOTCH1 expression accompanies the dysregulation of mitochondrial morphological dynamics in mice with diabetic cardiac fibrosis

To assess the role of NOTCH family members in diabetic cardiac fibrosis, we first established two mouse models of cardiac fibrosis in type 2 diabetes using leptin receptor-deficient mice (db/db) and mice fed a high-fat diet (HFD) with streptozotocin (STZ) (Fig. 1A). After one week of acclimatization, we measured weekly the fasting blood glucose values of mice in each group to ensure the successful establishment of diabetic cardiac fibrosis in mice (Figure S1A, S1E and S1K). Food and water intake and body weight were higher in these mouse models (Figure S1B–1D and S1F–1 H). Next, we used RT-qPCR and western blotting to screen for changes in the NOTCH family in the fibrotic heart tissues of diabetic mice. Only Notch1 expression was significantly decreased in the fibrotic tissue of the model group, whereas Notch2, Notch3, and Notch4 expression remained unchanged (Fig. 1B; Figure S1I). These data suggest that Notch1 expression is decreased in diabetic cardiac fibrosis.

Fig. 1
figure 1

Decreased NOTCH1 expression accompanies the dysregulation of mitochondrial morphological dynamics in mice with diabetic cardiac fibrosis. A Construction diagram of two animal models of diabetic cardiac fibrosis. B Western blotting analyzed the expression of Notch family in two animal models of diabetic cardiac fibrosis (two-tailed Student t test; n = 6). C Construction diagram of the animal model of diabetic cardiac fibrosis in Notch1 conditional knockout (cKO) mice with HFD/STZ. D Western blotting analyzed the expression of various markers related to mitochondrial fission, fusion, autophagy, and biogenesis in STZ + HFD + cKO-Notch1 model and control group (two-tailed Student t test; n = 6). E Mitochondrial morphodynamics of heart tissue in STZ + HFD + cKO-Notch1 mouse model observed by transmission electron microscopy (two-tailed Student t test; n = 6). F Confocal microscope images showing double staining results of POSTN (pseudo-colored light blue) and Mito Tracker in cardiac fibrotic tissue of STZ + HFD + cKO-Notch1 group and control group mice (two-tailed Student t test; n = 6). Scale bars: 10 μm. G Western blotting analyzed the expression of POSTN, Fibronectin, Collagen I, and Collagen III proteins in STZ + HFD + cKO-Notch1 model and control group (two-tailed Student t test; n = 6). H Confocal microscope images showing double staining results of POSTN (pseudo-colored light blue) and Notch1 in cardiac fibrotic tissue of STZ + HFD + cKO-Notch1 group and control group mice (two-tailed Student t test; n = 6). Scale bars: 10 μm. I Confocal microscope images showing triple staining results of POSTN (pseudo-colored light blue), CTnT (pseudo-colored yellow) and Mito Tracker in cardiac fibrotic tissue of STZ + HFD + cKO-Notch1 group and control group mice (two-tailed Student t test; n = 6). Scale bars: 10 μm and 20 μm

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Notch signaling has been reported to restore mitochondrial morphological dynamics [11], but little is known about the role of NOTCH1 in controlling these dynamics in diabetic cardiac fibrosis. We hypothesized that NOTCH1 regulates mitochondrial morphological dynamics in diabetic cardiac fibrosis. Using the HFD/STZ model, we established a mouse model of diabetic cardiac fibrosis with CF-specific Notch1 conditional knockout (POSTN-Cre× Notch1flox/flox, Notch1-cKO; Fig. 1C). To investigate changes in mitochondrial morphological dynamics in Notch1-cKO mice with diabetic cardiac fibrosis, we screened various markers related to mitochondrial fission, fusion, autophagy, and biogenesis. Western blotting showed a significant increase in the key mitochondrial fission marker DRP1 in the heart tissues of Notch1-cKO mice with diabetic cardiac fibrosis (Fig. 1D), and RT-qPCR confirmed these changes (Figure S1J). Additionally, mitochondrial fission markers (MID49), mitochondrial fusion markers (MFN1 and MFN2), autophagy markers (PINK1 and Parkin), and biogenesis markers (TFAM and NRF1) in the fibrotic tissues of these mice were assessed using Western blots (Fig. 1D) and RT-qPCR (Figure S1J). Notably, the levels of these markers did not exhibit significant changes.

This suggests that mitochondrial fission was responsible for the observed increase in mitochondrial fragmentation, but not in mitochondrial fusion, autophagy, or biogenesis, in Notch1-cKO mice with diabetic cardiac fibrosis. In further experiments assessing mitochondrial morphology in heart tissues from mouse models of diabetic cardiac fibrosis, transmission electron microscopy showed increased mitochondrial numbers, but decreased length and number of crests in mitochondria, in fibrotic cardiac tissues of Notch1-cKO mice (Fig. 1E). MitoTracker Red probes indicated a significantly higher proportion of mitochondria in the fibrotic tissues of the model group (Fig. 1F). These results demonstrated that Notch1-cKO promotes mitochondrial fission in diabetic cardiac fibrosis.

Next, we used echocardiography to monitor changes in heart function in Notch1-cKO mice with diabetic cardiac fibrosis. Echocardiography analysis showed impaired heart function in terms of significantly reduced fractional shortening and decreased ejection fraction in Notch1-cKO mice compared to wild-type (WT) mice (Figure S1L). The diastolic function tests E/e’ and E/A were also significantly decreased in Notch1-cKO mice with diabetic cardiac fibrosis, indicating impaired diastolic function (Figure S1L).

As increased cardiac fibrosis is closely associated with impaired heart function [22], we validated the presence of fibrosis using histopathology and measurement of fibrosis marker expression. Masson’s trichrome and Sirius Red stainings of heart tissue from Notch1-cKO mice with diabetic cardiac fibrosis showed significant increases in collagen deposition (Figure S1L), as well as increased expression of POSTN, fibronectin, collagen I, and collagen III (Fig. 1G). Interestingly, we observed decreased NOTCH1 staining in fibroblasts (co-staining of NOTCH1 and the fibroblast marker POSTN [23], Fig. 1H) but not in cardiomyocytes (co-staining of NOTCH1 and cardiac troponin T [cTnT], Figure S1M). Likewise, the MitoTracker Red probe exhibited synchronization with changes in the expression of the fibroblast marker POSTN but but changes in the cardiomyocyte marker cTnT in fibrotic heart tissue were not significan (Fig. 1I). Moreover, the MitoTracker Red probe was not colocalized with the endothelial cell marker CD31, smooth muscle cell marker α-SMA, macrophage marker CD68, and B cells marker CD79a in the heart tissue of Notch1-cKO mice with diabetic cardiac fibrosis (Figure S1N), indicating fibroblast-specific decreases in Notch1 expression accompanied by mitochondrial fission in diabetic cardiac fibrosis. These results provide further evidence for the link between fibroblast-specific decreases in NOTCH1 expression and excessive mitochondrial fission, indicating the potential impact of fibroblast-specific dysregulation of NOTCH1 on diabetic cardiac fibrosis.

Fibroblast-specific NOTCH1-NICD complementation inhibits mitochondrial fission and ameliorates diabetic cardiac fibrosis

Next, we investigated whether mitochondrial fission is similarly enhanced in vitro by differentiating primary CFs and cardiomyocytes isolated from neonatal mice (Fig. 2A). To mimic type 2 diabetes in vitro, we used high-glucose/high-fat (HG/HF) medium (33 mmol/L glucose, 200 µmol/L palmitate, and 200 µmol/L oleate). Interestingly, transmission electron microscopy assay found that decreased length of mitochondrial crests in the mitochondria of cardiac fibroblasts treated with HG/HF (Fig. 2B). To investigate mitochondrial fission in HG/HF-induced CFs and cardiomyocytes, we evaluated various mitochondrial fission, fusion, autophagy, and biogenesis markers using RT-qPCR and western blotting. HG/HF exposure induced a significant increase in key mitochondrial fission markers (DRP1 and MID49) at the protein level in CFs (Fig. 2C; Figure S2A) but not in cardiomyocytes; similar changes were found in RT-qPCRs (Figure S2B and S2C). The levels of mitochondrial fusion (MFN1 and MFN2), autophagy (PINK1 and Parkin), and biogenesis (TFAM and NRF1) markers were slightly changed in CFs following HG/HF exposure without reaching significance (Fig. 2C; Figure S2A). None of these increases were evident in cardiomyocytes following HG/HF exposure, and RT-qPCRs showed similar changes (Figure S2B and S2C). We observed that MitoTracker Red levels increased in parallel with changes in POSTN in fibroblasts, while neither cTnT nor MITO exhibited significant changes in cardiomyocytes (Fig. 2D; Figure S2D), indicating that mitochondrial fission was enhanced in HG/HF-treated CFs in vitro. In vivo imaging data demonstrated similar trends (Fig. 2C and D; Figure S2A).

Fig. 2
figure 2

Fibroblast-specific NOTCH1-NICD complementation inhibits mitochondrial fission and ameliorates diabetic cardiac fibrosis. A Schematic diagram of a cell model simulating type 2 diabetes in vitro. B Mitochondrial morphodynamics in myocardial fibroblasts treated with HG/HF were observed using transmission electron microscopy (two-tailed Student’s t-test; n = 6). C Western blotting analyzed the expression of various markers related to mitochondrial fission, fusion, autophagy, and biogenesis in cardiac fibroblasts treated with HG/HF (two-tailed Student t test; n = 6). D Confocal microscope images showing the changes of double staining results of POSTN (pseudo-colored light blue) and Mito Tracker in cardiac fibroblasts treated with HG/HF (two-tailed Student t test; n = 6). Scale bars: 10 μm and 50 μm. E Western Blotting analyzed the changes of Notch1 family expression in cardiac fibroblasts treated with HG/HF (two-tailed Student t test; n = 6). F Confocal microscope images showing the changes of double staining results of POSTN (pseudo-colored light blue) and Notch1 in cardiac fibroblasts treated with HG/HF (two-tailed Student t test; n = 6). Scale bars: 10 μm and 50 μm. G Confocal microscope images showed changes in mitochondrial fission (pseudo-colored light blue) in HGHF-treated cardiac fibroblasts after Notch1 knockout (two-tailed Student t test; n = 6). Scale bars: 10 μm and 50 μm. H Using Edu assay to detect the proliferation ability of HGHF-treated cardiac fibroblasts after notch1 knockout (two-tailed Student t test; n = 6; Scale bars: 50 μm); The effect of Notch1 knockdown on HGHF-treated cardiac fibroblasts migration and invasion was determined by transwell assays (two-tailed Student t test; n = 6; Scale bars: 20 μm). I Masson trichromatic staining and Sirius red staining were performed on the cardiac fibrosis tissue sections of Notch1-cKO mice or db/db mice injected with AAV9-Postn-oeNotch1-NICD (two-tailed Student t test; n = 6). J–K: Confocal microscope images showed changes in mitochondrial fission in cardiac fibrosis tissue sections of Notch1-cKO mice or db/db mice injected with AAV9-Postn-oeNotch1-NICD (two-tailed Student t test; n = 6). Scale bars: 10 μm

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Using primary cultures of CFs and cardiomyocytes, we also investigated whether the expression of NOTCH family members was altered in vitro as in the abovementioned experiments. HG/HF treatment of CFs resulted in decreased levels of NOTCH1, whereas those of NOTCH2, NOTCH3, and NOTCH4 remained unchanged (Fig. 2E), and increased expression of POSTN and collagen I (Fig. 2E); none of these changes were evident in cardiomyocytes (Figure S2E). Moreover, we observed in HG/HF-induced CFs decreased staining for NOTCH1 (Fig. 2F); however, such changes were not evident in cardiomyocytes (Figure S2F). Notch1 knockdown enhanced mitochondrial fission in HG/HF-treated CFs, as demonstrated by fluorescence analysis (Fig. 2G). In our study, HG/HF treatment led to CF proliferation, migration, autophagy, apoptosis, differentiation, and pyroptosis. We observed that Notch1 knockdown significantly decreased CF proliferation and migration (Fig. 2H) but did not affect autophagy (BECN1 and P62), apoptosis (Bax and BCL-2), and pyroptosis (NLRP3 and GSDMD) markers (Figure S2G). These results indicated that Notch1 deletion was responsible for mitochondrial fission in and proliferation and migration of HG/HF-treated CFs.

According to our in vivo and in vitro results, Notch1 deletion promoted mitochondrial fission in diabetic cardiac fibrosis as well as CF proliferation and migration. To further confirm the role of fibroblast-specific NOTCH1-NICD complementation in Notch1-cKO mice with diabetic cardiac fibrosis, mice were injected with adeno-associated virus (AAV)9-Postn-oeNotch1-NICD to induce fibroblast-specific overexpression of Notch1-NICD (Figure S2H). NOTCH1-NICD expression was increased in the heart tissues of mice treated with AAV9-Postn-oeNotch1-NICD (Figure S2I). Notably, Notch1-NICD overexpression substantially decreased the expression levels of proliferating cell nuclear antigen (PCNA) and collagen I in fibrotic heart tissues from Notch1-cKO mice treated with AAV9-Postn-oeNotch1-NICD compared to those treated with AAV9-Postn-NC (Figure S2I). Consistent with these results, Masson’s trichrome and Sirius Red stainings showed that overexpression of Notch1-NICD markedly reduced collagen deposition and fibrosis in fibrotic heart tissues from Notch1-cKO mice treated with AAV9-Postn-oeNotch1-NICD compared to those treated with AAV9-Postn-NC (Fig. 2I). Echocardiography demonstrated in Notch1-cKO mice treated with AAV9-Postn-oeNotch1-NICD a further amelioration of heart dysfunction (Figure S2J). Moreover, a significant decrease in mitochondrial fission was observed in Notch1-cKO mice treated with AAV9-Postn-oeNotch1-NICD compared to those treated with AAV9-Postn-NC (Fig. 2J). Similar results were found in db/db mice treated with AAV9-Postn-oeNotch1-NICD (Fig. 2I and K; Figure S2I and S2J). Collectively, these findings suggest that fibroblast-specific NOTCH1-NICD complementation may underlie the inhibition of mitochondrial fission and the amelioration of diabetic cardiac fibrosis.

Notch1 modulates Drp1 expression and mitochondrial fission through transcriptional modulation

As Notch1 deletion facilitates mitochondrial fission [24] and fibroblast proliferation and migration [10], we hypothesized that NOTCH1-mediated mitochondrial fission would be necessary for the proliferation and migration of CFs. To test this hypothesis, we overexpressed the Notch1-NICD plasmid or knocked down Notch1 in CFs (Fig. 3A), which led to increased or decreased NOTCH1-NICD expression in CFs, respectively (Fig. 3B; Figure S3A). Super-resolution confocal microscopy showed significantly decreased mitochondrial fission in CFs overexpressing Notch1-NICD compared to those transfected with the vector, whereas Notch1 knockdown resulted in the opposite results (Figs. 2G and 3C), indicating that Notch1 knockdown promoted mitochondrial fission in CFs, whereas Notch1-NICD overexpression inhibited it.

Fig. 3
figure 3

Notch1 modulates Drp1 expression and mitochondrial fission through transcriptional modulation. A Schematic diagram of cardiac fibroblasts treated with either Notch1-NICD plasmid overexpression or Notch1 knockdown. B Western blotting verified the overexpression of NICD in HGHF-treated cardiac fibroblasts (two-tailed Student t test; n = 6). C Confocal microscope images showing the changes of double staining results of POSTN (pseudo-colored light blue) and Mito Tracker in cardiac fibroblasts treated with HG/HF after the overexpression of NICD (two-tailed Student t test; n = 6). Scale bars: 50 μm and 10 μm. D The expression of Hes1 and Hey1 in cardiac fibroblasts treated with HG/HF after the overexpression of NICD were analyzed by RT-qPCR (two-tailed Student t test; n = 6). E Western blotting analyzed the expression of various markers related to mitochondrial fission in HGHF-treated cardiac fibroblasts after notch1 knockout (two-tailed Student t test; n = 6). F Dual luciferase assay on 3T3 fibroblasts cotransfected with firefly luciferase constructs containing the Drp1 promoter and si-Notch1 or NICD plasmid (two-tailed Student t test; n = 6). G Confocal microscope images showing the changes of double staining results of Drp1 (pseudo-colored light blue) and Mito Tracker in Notch1 knockdown cardiac fibroblasts after Mdivi-1-treated (two-tailed Student t test; n = 6). Scale bars: 50 μm. H ChIP experiment analyzed the binding of NICD to the Drp1 promoter region, n = 3. I CO-IP experiment analyzed the binding of Notch1-NICD and Drp1 associated transcriptional cofactor, include Zeb1, YAP, YY2, and NR4A1, n = 3. J CO-IP experiment analyzed the binding of NICD and ZEB1 in the HG/HF environment, n = 3. K ChIP experiment analyzed the binding of ZEB1 to the Drp1 promoter region in the HG/HF environment, n = 3. L ChIP-qPCR experiment analyzed the binding of ZEB1 to the Drp1 promoter region in Notch1 knockdown treated and HG/HF-treated cardiac fibroblasts (two-tailed Student t test; n = 6). M A dual luciferase assay was performed on 3T3 fibroblasts co-transfected with firefly luciferase constructs containing the Drp1 promoter and vectors expressing either ZEB1, NICD, or both (two-tailed Student t test; n = 6)

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Because the classical NOTCH1 pathway molecules HES1 and HEY1, showed marked changes (Fig. 3D), we hypothesized that NOTCH1 would modulate mitochondrial fission through transcriptional regulation. Notch1 knockdown significantly decreased DRP1 expression (Fig. 3E) but did not affect MFF, MID49, MID51, or FIS1 expression (Fig. 3E). Similar changes were found in RT-qPCRs (Figure S3B). To further validate our findings regarding the correlation between Notch1 and Drp1 activity, we initially confirmed that the Drp1 luciferase reporter efficiently showed Drp1 transcriptional activity (Fig. 3F). Notch1 knockdown significantly increased Drp1 transcriptional activity, whereas Notch1-NICD overexpression had the opposite effect (Fig. 3F). Exposure to the classical DRP1 inhibitor mitochondrial division inhibitor 1 (mdivi-1) decreased Drp1 expression in Notch1-knockdown fibroblasts. Moreover, mdivi-1 exposure abrogated the effect of Notch1 on Drp1 expression (Fig. 3G; Figure S3C). Chromatin immunoprecipitation (ChIP)-PCR revealed that NOTCH1-NICD was bound to the promoter region of Drp1 and was involved in the transcriptional activation of Drp1 (Fig. 3H). Next, we investigated how NOTCH1-NICD regulates the transcription of Drp1. NOTCH1-NICD may participate in Drp1 transcription via the activation or inhibition of transcriptional cofactors including zinc finger E-box-binding homeobox 1 (Zeb1) [25], Yap [26], Yy2 [27], and Nr4a1 [28]. Therefore, we explored whether NOTCH1-NICD recruits these cofactors to regulate Drp1 transcription in CFs. Co-immunoprecipitation assays showed that the transcription corepressor Zeb1, but not Yap, Yy2, or Nr4a1, interacted with NOTCH1-NICD in CFs (Fig. 3I). Moreover, this assay showed that Zeb1 pulled down by the NOTCH1-NICD antibody was significantly reduced in HG/HF-induced CFs (Fig. 3J), indicating that Notch1 deficiency in the HG/HF environment might reduce the recruitment of the corepressor Zeb1, leading to increased Drp1 transcriptional activity and increased DRP1 expression. Next, we performed ChIP experiments using anti-ZEB1 antibodies. Under HG/HF conditions, Zeb1 localization to the same Drp1 promoter region as Notch1-NICD, was significantly reduced, thereby decreasing the downregulation of Drp1 transcription (Fig. 3K and L), similar to the results of the luciferase reporter assay (Fig. 3M).

In agreement with these results, Drp1 knockdown abrogated the effects of Notch1 deletion on the promotion of CF proliferation and migration (Figure S3D), and similar results were found in mdivi-1-treated CFs (Figure S3E). Collectively, these data suggest that the loss of Notch1 promotes Drp1 transcription, mitochondrial fission, and CF proliferation and migration.

Decreases in NOTCH1 expression and mitochondrial fission are caused by ALKBH5-dependent increases in m6A methylation of Notch1 mRNA

Next, we explored the mechanisms underlying NOTCH1 downregulation in diabetic fibrotic heart tissues and HG/HF-induced CFs. Recent studies indicate that m6A exerts both stimulatory and inhibitory effects on translation dynamics. The precise impact of m6A on translation appears to depend on its specific locations of the transcript [29]. Using SRAMP software (http://www.cuilab.cn/sramp) and RMBase (https://rna.sysu.edu.cn/rmbase/), we found a strong m6A peak enrichment of Notch1 in mice (Fig. 4A), indicating that Notch1 can be regulated by m6A methylation. The expression of genes associated with m6A modifications was investigated, and our results were consistent with the bioinformatic findings. Both HG/HF-treated fibroblasts in vitro and heart tissues of mice with diabetic cardiac fibrosis in vivo exhibited marked increases in METTL3, METTL14, and WTAP expression, whereas a marked decrease in ALKBH5 and a slight increase in fat mass and obesity-associated (FTO) expression were observed (Fig. 4B C; Figure S4A and 4B). In cardiomyocytes, no significant changes in the corresponding expression levels were observed (Figure S4C). Both in vitro and in vivo tests demonstrated significant increases in m6A mRNA levels of the model groups compared to those of the control groups (Fig. 4D and E).

Fig. 4
figure 4

Decreases in NOTCH1 expression and mitochondrial fission are caused by ALKBH5-dependent increases in m6A methylation of Notch1 mRNA. A m6A peak enrichment of Notch1 predicted by SRAMP software and RMBase. B Western blotting analyzed the changes of m6A modification enzyme expression in HG/HF treated cardiac fibroblasts (two-tailed Student t test; n = 6). C Western blotting analyzed the expression of m6A modifying enzyme in diabetic cardiac fibrosis tissue of db/db mice (two-tailed Student t test; n = 6). D–E The changes of m6A levels in the genome of type 2 diabetes models in vitro and in vivo were detected by dot blotting (two-tailed Student t test; n = 6). F Western blotting analyzed the changes of Notch1 expression after ALKBH5 overexpression in HGHF-treated cardiac fibroblasts (two-tailed Student t test; n = 6). G The changes of m6A levels in the in HGHF-treated cardiac fibroblasts after ALKBH5 overexpression were detected by dot blotting (two-tailed Student t test; n = 6). H The stability of Notch1 transcripts in HG/HF-treated and ALKBH5-overexpressing cardiac fibroblasts, post-ActD treatment, was assessed by RT-qPCR (two-tailed Student t test; n = 3). I MeRIP-qPCR analyzed the changes of m6A levels at three different sites of Notch1 mRNA from HG/HF-treated cardiac fibroblasts after ALKBH5 overexpression (two-tailed Student t test; n = 6). J Schematic illustration of a point mutation at m6A-site 1 of Notch1 mRNA. K RIP analysis revealed the altered interaction between ALKBH5 and Notch1 in HG/HF-treated cardiac fibroblasts following ALKBH5 overexpression (two-tailed Student t test; n = 6). L RNA pull-down assays were performed to assess the binding capability of wild-type and mutant Notch1 to ALKBH5, followed by western blot analysis using an anti-ALKBH5 antibody (two-tailed Student t test; n = 3). M RIP analysis revealed the altered interaction between ALKBH5 and wild-type or mutant Notch1 (two-tailed Student t test; n = 6). N The changes of m6A level of mutant Notch1 mRNA after ALKBH5 overexpression in cardiac fibroblasts were analyzed by MeRIP-qPCR (two-tailed Student t test; n = 6). O Confocal microscope images showed changes in mitochondrial fission (pseudo-colored light blue) in HGHF-treated cardiac fibroblasts following ALKBH5 overexpression (two-tailed Student t test; n = 6). Scale bars: 50 μm and 10 μm

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Based on these results, we hypothesized that ALKBH5, METTL3, METTL14, and WTAP regulate NOTCH1 expression. The successful overexpression of Alkbh5 and knockdown of Mettl3, Mettl14, and Wtap were validated at both protein and mRNA levels (Fig. 4F; Figure S4D–4F). Surprisingly, only Alkbh5 overexpression significantly increased NOTCH1 expression, whereas knockdown of Mettl3, Mettl14, and Wtap did not affect NOTCH1 expression (Fig. 4F; Figure S4D–4F). Correspondingly, Alkbh5 overexpression significantly decreased m6A mRNA levels in CFs (Fig. 4G). Sequence analysis revealed three matches of common m6A sequences in Notch1 (Fig. 4A; Table S4). An RNA decay assay showed that Alkbh5 overexpression remarkably increased the stability of Notch1 mRNA, whereas knockdown of ALKBH5 reduced Notch1 mRNA stability (Fig. 4H; Figure S4G). m6A-methylated RNA immunoprecipitation (MeRIP)-qPCR confirmed the significant m6A-mediated reduction of Notch1 expression following Alkbh5 overexpression in HG/HF-treated CFs compared to that in IgG- immunoprecipitated control CFs (Fig. 4I). Based on these MeRIP-qPCR results, we mutated site 1 in Notch1 (Notch1-mut; Fig. 4J; Table S2). Alkbh5 overexpression enhanced the enrichment of the WT Notch1 reporter (Fig. 4K). The specificity of the association between Alkbh5 and Notch1 was further confirmed by RNA pull-down and immunoprecipitation assays (Fig. 4L and M). A MeRIP assay confirmed that Notch1-mut enrichment was not increased by Alkbh5 overexpression (Fig. 4N).

Previous studies have reported that ALKBH5 is involved in RNA methylation, cell proliferation, and migration [30]. As expected, Alkbh5 overexpression significantly decreased the proliferation rate and migration of CFs (Figure S4H). Moreover, Alkbh5 overexpression reduced mitochondrial fission (Fig. 4O) and downregulated the expression of PCNA and DRP1 (Figure S4I) in CFs. Collectively, these results indicate that mitochondrial fission and NOTCH1 expression decrease due to enhanced N6-adenine methylation of Notch1 mRNA in an ALKBH5-dependent manner.

Increased m6A of Notch1 mRNA Enhances YTHDF2 phase separation and subsequent binding of YTHDF2 to Notch1 mRNA, thereby increasing its degradation

Using m6A dot blotting, we showed that Alkbh5 knockdown significantly increased m6A levels (Fig. 5A). As the m6A status is dominated by the activity of m6A readers [31], we identified the reader protein most closely related to m6A-mediated Notch1 modification and investigated m6A reader-associated gene expression (Fig. 5B). YTHDF1 and YTHDF2 expression was markedly enhanced in HG/HF-induced CFs and murine fibrotic heart tissue (Fig. 5C and D; Figure S5A and S5B), whereas YTHDF3, IGF2BP1 (Fig. 5C and D; Figure S5A and S5B), YTHDC1, YTHDC2, IGF2BP2, and IGF2BP3 (Figure S5A and S5B) expression levels were not significantly increased in the same groups. Cardiomyocytes that received similar treatments showed no significant changes in expression levels (Figure S5C). Thus, we hypothesized that YTHDF1 and YTHDF2 would regulate Notch1 expression. Successful knockdown of YTHDF1 and YTHDF2 was validated at both mRNA and protein levels (Fig. 5E and F; Figure S5D and S5E). Ythdf2 knockdown significantly increased Notch1 expression, whereas Ythdf1 knockdown did not change Notch1 expression (Fig. 5E and F), suggesting that Notch1 expression is posttranscriptionally regulated by Ythdf2.

Fig. 5
figure 5

Increased m6A of Notch1 mRNA enhances YTHDF2 phase separation and subsequent binding of YTHDF2 to Notch1 mRNA, thereby increasing its degradation. A The changes of m6A levels in cardiac fibroblasts after Notch1 knockout were detected by dot blotting (two-tailed Student t test; n = 6). B The PPI network performed proteomic analysis of the reader (n = 14) most closely associated with m6A -mediated Notch1 modification. C Western blotting analyzed the changes of m6A readers-associated gene expression in HG/HF treated cardiac fibroblasts (two-tailed Student t test; n = 6). D Western blotting analyzed the changes of m6A readers-associated gene expression in diabetic cardiac fibrosis tissue of db/db mice (two-tailed Student t test; n = 6). E– F Western blotting analyzed the changes of notch1 expression after YTHDF1/2 knockout in HGHF-treated cardiac fibroblasts (two-tailed Student t test; n = 6). G The stability of Notch1 transcripts in HG/HF-treated and YTHDF2-knockout cardiac fibroblasts, post-ActD treatment, was assessed by RT-qPCR (two-tailed Student t test; n = 3). H RIP analysis revealed the altered interaction between ALKBH5 and Notch1 in HG/HF-treated cardiac fibroblasts following YTHDF2 knockout (two-tailed Student t test; n = 6). I Confocal microscope images showing the distribution of YTHDF2 (pseudo-colored yellow) in the CFs. Scale bars: 10 μm. J Representative fluorescence microscopy images in vitro phase separation assay of YTHDF2 following treatment with 1,6-hexanediol (1,6-HD) (n = 3). Scale bars: 50 μm. K Representative fluorescence microscopy images in vitro phase separation assay on YTHDF2 following treatment with m6A nucleotides or non-m6A nucleotides (n = 3). Scale bars: 50 μm. L The stability of Notch1 transcripts in HG/HF-treated and 1,6-hexanediol-treated cardiac fibroblasts, post-ActD treatment, was assessed by RT-qPCR (two-tailed Student t test; n = 3). M Western blotting analyzed the expression changes of YTHDF2, Notch1, and Drp1 proteins in HGHF-treated cardiac fibroblasts following 1,6-HD treatment (two-tailed Student t test; n = 6). N Confocal microscope images showed changes in mitochondrial fission (pseudo-colored light blue) in HGHF-treated cardiac fibroblasts following 1,6-HD treatment (two-tailed Student t test; n = 6). Scale bars: 50 μm and 10 μm

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Results of RNA stability assays showed that the stability of Notch1 mRNA increased in CFs treated with siRNA-Ythdf2 (Fig. 5G). RIP-qPCR confirmed the presence of m6A methylation in Notch1, which was recognized by Ythdf2. Moreover, Notch1 enrichment was significantly decreased upon Ythdf2 knockdown (Fig. 5H). Based on the MeRIP-qPCR results, we mutated the site 1 in Notch1 (Notch1-mut; Figure S5F). The specificity of the association between Ythdf2 and Notch1 was further confirmed by RNA pull-down and RNA immunoprecipitation assays; Ythdf2 was bound to site 1 in Notch1 but not to Notch1-mut (Figure S5G and S5H). According to the RIP assay, Notch1-mut enrichment was not altered by Ythdf2 knockdown (Figure S5I).

Interestingly, abnormal accumulation of YTHDF2 in HG/HF-induced CFs was observed (Fig. 5I). Various RNA-binding proteins undergo liquid-liquid phase separation, forming liquid-like droplets and regulating the fate of mRNAs [18]. To understand how YTHDF2 affects the fate of m6A mRNA, we hypothesized that m6A-modified mRNAs are governed by liquid-liquid phase separation and examined the properties of YTHDF2 condensation in CFs. We purified full-length recombinant YTHDF2 fused to GFP expressed in E. coli cells and performed phase separation assays in vitro. Fluorescence recovery after photobleaching assays demonstrated that the fluorescence signal of the YTHDF2-GFP protein recovered after bleaching (Figure S5J). Moreover, treatment with 1,6-hexanediol [32], an aliphatic alcohol, inhibited the weak hydrophobic protein-protein/protein-RNA interactions required for droplet formation (droplet melting activity) and significantly disrupted the droplet formation of YTHDF2 (Fig. 5J). To examine the effects of m6A modification on YTHDF2 phase separation, we analyzed the changes in droplet formation of YTHDF2 in the presence of m6A nucleotides. Interestingly, m6A nucleotides triggered droplet formation in YTHDF2, and in vitro phase separation assays showed that m6A-containing RNA enhanced the phase separation of YTHDF2 (Fig. 5K).

In CFs treated with 1,6-hexanediol, Notch1 mRNA stability was increased (Fig. 5L), Notch1 expression was upregulated, Drp1 expression was downregulated (Fig. 5M), and mitochondrial fission was reduced (Fig. 5N). Ythdf2 knockdown markedly reduced the proliferation and migration of CFs (Figure S5K). Additionally, Ythdf2 knockdown reduced mitochondrial fission (Figure S5L) and suppressed PCNA and DRP1 expression in CFs (Figure S5M). Notably, when Ythdf2 was knocked down, Notch1 knockdown restored Drp1 levels, whereas Ythdf2 overexpression mitigated the downregulation of Drp1 caused by Notch1 overexpression (Figure S5N and S5O). Collectively, these data demonstrate that increased m6A of Notch1 mRNA enhances YTHDF2 phase separation and subsequent binding of YTHDF2 to Notch1 mRNA, thereby increasing its degradation.

Rescuing NOTCH1 expression by epitranscriptomic downregulation ameliorates diabetic cardiac fibrosis

To determine the role of CF-specific Alkbh5 overexpression or Ythdf2 knockdown in diabetic cardiac fibrosis, we subcutaneously injected 16-week-old diabetic cardiac fibrosis mouse models [33] and 13-week-old diabetic mouse models with AAV9-Postn-oeAlkbh5 or AAV9-Postn-shYthdf2 (Fig. 6A). The blood glucose levels did not differ between the treatment and control groups (Figure S6A and S6B). Compared with AAV9-NC-treated mice with diabetic cardiac fibrosis, ALKBH5 expression was markedly increased in AAV9-Postn-oeAlkbh5-treated mice whereas YTHDF2 expression was significantly decreased in AAV9-Postn-shYthdf2-treated mice (Fig. 6B C) Both Alkbh5 overexpression with AAV9-Postn-oeAlkbh5 and Ythdf2 knockdown with AAV9-Postn-shYthdf2 substantially reduced the levels of POSTN, PCNA, and collagen I (Fig. 6B C). In accordance with these findings, Masson’s trichrome and Sirius Red stainings showed that both AAV9-Postn-oeAlkbh5- and AAV9-Postn-shYthdf2-treated mice with diabetic cardiac fibrosis showed marked reductions in collagen deposition and fibrosis (Figure S6C). In contrast, the single diabetic mouse model showed no significant fibrosis collagen deposition (Figure S6D). Using echocardiography to evaluate AAV9-Postn-oeAlkbh5- and AAV9-Postn-shYthdf2-treated mice with diabetic cardiac fibrosis, we observed that heart dysfunction was ameliorated in these model groups (Figure S6E). There was no significant change of heart function in the single diabetic mouse model (Figure S6F).

Fig. 6
figure 6

Rescuing NOTCH1 expression by epitranscriptomic downregulation ameliorates diabetic cardiac fibrosis. A Schematic diagram of animal models with adeno-associated virus targeting cardiac fibroblasts, injected via the tail vein in db/db mice. B–C Western blotting analyzed the expression changes of ALKBH5/YTHDF2, Collagen I, POSTN, PCNA, Notch1, and Drp1 proteins in cardiac fibrosis tissue of db/db mice injected with AAV9-Postn-oeALKBH5 or AAV9-Postn-shYTHDF2 (two-tailed Student t test; n = 6). D–E: Confocal microscope images showed changes in mitochondrial fission in cardiac fibrosis tissue sections of db/db mice injected with AAV9-Postn-oeALKBH5 or AAV9-Postn-shYTHDF2 (two-tailed Student t test; n = 6). Scale bars: 10 μm. F–G: Confocal microscope images showing double staining results of Notch1 and Drp1 (pseudo-colored light blue) in cardiac fibrosis tissue sections of db/db mice injected with AAV9-Postn-oeALKBH5 or AAV9-Postn-shYTHDF2 (two-tailed Student t test; n = 6). Scale bars: 20 μm and 10 μm. H: The changes of m6A levels in cardiac fibrosis tissue of db/db mice injected with AAV9-Postn-oeALKBH5 were detected by dot blotting (two-tailed Student t test; n = 6). I: MeRIP-qPCR analyzed the changes of m6A levels in cardiac fibrosis tissue of db/db mice injected with AAV9-Postn-oeALKBH5 (two-tailed Student t test; n = 6). J: RIP analysis revealed the altered interaction between YTHDF2 and Notch1 in cardiac fibrosis tissue of db/db mice injected with AAV9-Postn-shYTHDF2 (two-tailed Student t test; n = 6). K–L: Confocal microscope images showed changes in mitochondrial fission in cardiac fibrosis tissue sections of ALKBH5 overexpression or YTHDF2 knockdown in diabetic cardiac fibrosis mice following Notch1-knockdown (two-tailed Student t test; n = 6). Scale bars: 10 μm

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Furthermore, we observed decreased mitochondrial fragmentation and longer mitochondria in mice with diabetic cardiac fibrosis following Alkbh5 overexpression or Ythdf2 knockdown (Fig. 6D and E). Moreover, the expression of Notch1 was increased and that of Drp1 decreased in AAV9-Postn-oeAlkbh5- and AAV9-Postn-shYthdf2-treated mice compared to AAV9-NC-treated mice (Fig. 6B and C). Similar changes were observed in immunofluorescence stainings (Fig. 6F and G). Following AAV9-Postn-oeAlkbh5 treatment of mice with diabetic cardiac fibrosis, we also observed the downregulation of m6A levels (Fig. 6H). MeRIP-qPCRs confirmed the m6A-mediated significant reduction of Notch1 in AAV9-Postn-oeAlkbh5-treated mice compared to the IgG group (Fig. 6I), whereas the enrichment of Notch1 was significantly decreased upon Ythdf2 knockdown (Fig. 6J).

To verify in vivo whether Alkbh5 and Ythdf2 regulate diabetic cardiac fibrosis by targeting Notch1, we knocked down Notch1 following Alkbh5 overexpression or Ythdf2 knockdown in mice with diabetic cardiac fibrosis. Masson’s trichrome and Sirius Red stainings revealed that compared with Alkbh5 overexpression alone or Ythdf2 knockdown alone, Notch1 knockdown with Alkbh5 overexpression or Ythdf2 knockdown resulted in increased collagen deposition (Figure S6G and S6H), enhanced mitochondrial fission (Fig. 6K and L), and impaired heart function (Figure S6I and S6J) in the model groups. Collectively, these results suggest that rescuing Notch1 expression by epitranscriptomic downregulation ameliorates diabetic cardiac fibrosis.

In patients with DCM, NOTCH1 expression is decreased, mitochondrial fission enhanced, ALKBH5 expression downregulated, and YTHDF2 expression upregulated

Finally, we examined CF-, collagen-, and mitochondrial fission-related markers, m6A levels, and ALKBH5, YTHDF2, and NOTCH1 levels in patients with DCM. The m6A levels in fibrotic heart tissues from patients with DCM were significantly higher than those from controls (Fig. 7A). Consistent with these increased m6A levels, NOTCH1 expression in heart tissues of patients with DCM was decreased compared to that of controls (Fig. 7B). Moreover, mitochondrial fragmentation was significantly increased, and mitochondria were shorter in samples from patients with DCM than in those from controls (Fig. 7C; Figure S7A). Compared with heart tissues from controls, colocalization of Mito Tracker Red and POSTN was significantly increased in cardiac tissues from patients with DCM (Fig. 7D; Figure S7B), as was the expression of the mitochondrial fission marker DRP1 (Fig. 7B), indicating enhanced mitochondrial fission in the heart tissues of patients with DCM. Moreover, ALKBH5 expression was downregulated and YTHDF2 expression was upregulated in patients with DCM, in agreement with the results of the immunofluorescence assays (Fig. 7E; Figure S7C). Consistent with these observations, RIP analysis showed that the enrichment of Notch1 to Alkbh5 significantly decreased, but the enrichment of Notch1 to Ythdf2 markedly increased in the heart tissues of patients with DCM (Fig. 7F and G). Accordingly, Masson’s trichrome and Sirius Red stainings revealed thick and highly disorganized collagen in the heart tissues of patients with DCM (Fig. 7H), and mRNA and protein expression levels of the fibrosis markers collagen I and PCNA were increased in these patients (Fig. 7B). Furthermore, heart function was impaired in patients with DCM, as evidenced by a significantly reduced fractional shortening and decreased ejection fraction (Table S1). Moreover, we observed decreased NOTCH1 staining in the fibroblasts of fibrotic hearts by co-staining NOTCH1 and the fibroblast marker POSTN (Fig. 7I; Figure S7D). These data confirm that in patients with DCM, NOTCH1 expression is decreased, accompanied by enhanced mitochondrial fission, downregulation of ALKBH5 expression, and upregulation of YTHDF2 expression.

Fig. 7
figure 7

In patients with DCM, NOTCH1 expression is decreased, mitochondrial fission enhanced, ALKBH5 expression downregulated, and YTHDF2 expression upregulated. A The changes of m6A levels in the fibrotic heart tissues of patients with DCM were detected by dot blotting (two-tailed Student t test; n = 6). B Western blotting analyzed the expression changes of Notch1, Drp1, ALKBH5, YTHDF2, Collagen I, POSTN, and PCNA proteins in the fibrotic heart tissues of patients with DCM (two-tailed Student t test; n = 6). C Confocal microscope images showing double staining results of Mito Tracker and Drp1 (pseudo-colored light blue) in the fibrotic heart tissue sections of patients with DCM (two-tailed Student t test; n = 6). Scale bars: 20 μm. D Confocal microscope images showing double staining results of Mito Tracker and POSTN (pseudo-colored light blue) in the fibrotic heart tissue sections of patients with DCM (two-tailed Student t test; n = 6). Scale bars: 20 μm. E Confocal microscope images showing double staining results of ALKBH5 and YTHDF2 (pseudo-colored light blue) in the fibrotic heart tissue sections of patients with DCM (two-tailed Student t test; n = 6). Scale bars: 20 μm. F–G: RIP analysis revealed the altered interaction between ALKBH5/YTHDF2 and Notch1 in the fibrotic heart tissues of patients with DCM (two-tailed Student t test; n = 6). H Masson trichromatic staining and Sirius red staining were performed in the fibrotic heart tissue sections of patients with DCM (two-tailed Student t test; n = 6). I Confocal microscope images showing double staining results of Notch1 and POSTN (pseudo-colored light blue) in the fibrotic heart tissue sections of patients with DCM (two-tailed Student t test; n = 6). Scale bars: 20 μm

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Discussion

Although the molecular targets and treatments for diabetic cardiac fibrosis have been extensively studied over the past few decades, the condition cannot be reversed or sufficiently alleviated [34]. The resulting decline in diastolic and subsequent systolic function presents a significant challenge to the prognosis of patients with diabetic cardiomyopathy [35]. The pathogenesis of diabetic cardiomyopathy is complex and multifactorial, and the specific molecular mechanisms underlying diabetic cardiac fibrosis remain unclear. This study demonstrated that Notch1 deficiency is accompanied by excessive mitochondrial fission in models of diabetic cardiac fibrosis and patients with DCM. We comprehensively demonstrated that rescuing fibroblast-specific Notch1 expression ameliorated diabetic cardiac fibrosis via epitranscriptomic downregulation. Mechanistically, Notch1 downregulation was associated with Alkbh5-mediated m6A demethylation in the 3’UTR of Notch1 mRNA and elevated m6A mRNA levels. This markedly enhanced YTHDF2 phase separation increased the recognition of m6A residues in Notch1 mRNA by YTHDF2, and induced Notch1 degradation. Furthermore, we identified a novel epitranscriptomic mechanism for Alkbh5 and Ythdf2 as gatekeeper regulators of Notch1 expression, promoting mitochondrial fission and aggravating diabetic cardiac fibrosis (Fig. 8).

Fig. 8
figure 8

A novel epitranscriptomic mechanism for Alkbh5 and Ythdf2 as gatekeeper regulators of Notch1 expression, promoting mitochondrial fission and aggravating diabetic cardiac fibrosis

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The NOTCH pathway has been implicated in the development of fibrosis across various organs, and recent studies have revealed a functional antagonism between N1ICD and Smad3 in relation to the cardiac fibroblasts phenotype [36, 37]. However, data regarding its role in diabetic cardiac fibrosis remain limited. Our findings suggest a protective role of Notch1 against collagen production and fibrosis in DCM. In contrast to previous studies that directly limited Notch1 to enhance mitochondrial fission and fibroblast proliferation, our findings suggest that increased Notch1 expression via epitranscriptomic downregulation decreases mitochondrial fission in models of diabetic cardiac fibrosis and patients with DCM. Previously, we reported that limiting mitochondrial fission attenuates cardiac fibrosis by limiting CF proliferation [21]. Since CF proliferation is an established mediator of fibrosis, we speculated that Notch1 might play a role in fibrosis. Herein, we introduce a new mechanism by which Notch1 upregulation via epitranscriptomic downregulation ameliorates diabetic cardiac fibrosis. These findings provide new mechanistic insights into Notch1 functions in DCM and identify multiple novel strategies to directly or indirectly promote Notch1 expression as a potential therapeutic approach to restrict diabetic cardiac fibrosis in DCM.

It is worth mentioning that the main study exploring the role of NOTCH1 in fibrosis focused on the potential antifibrotic effects of NOTCH1 pathway activation [38], with limited data on Notch1 regulation by epitranscriptomics. More recently, pulmonary fibrosis was associated with the induction of NOTCH family proteins, such as NOTCH1 [39] and NOTCH3 [40]. However, NOTCH1 expression in DCM, and the roles of mitochondrial fission and NOTCH1 in diabetic cardiac fibrosis remain unexplored.

Herein, we found that mRNA and protein levels of NOTCH1 were decreased in fibrotic cardiac tissues of patients with DCM. Consistent with our mouse model of diabetic cardiac fibrosis, cardiac NOTCH1 levels positively correlated with impaired heart function. NOTCH1 protein and mRNA levels were decreased in the hearts of mice with diabetic cardiac fibrosis, suggesting that cardiac NOTCH1 expression might be inhibited at the transcriptional or posttranscriptional level. Our data suggest that the posttranscriptional effects of NOTCH1 are significantly correlated with its epitranscriptomic characteristics. Further studies exploring the epitranscriptomic regulation of NOTCH1 in diabetic cardiac fibrosis are required.

Although epitranscriptomic mechanisms regulating cardiac function and cardiac fibrosis-related diseases are understood to some extent [41], more explorations are needed to reveal the specific molecular targets of epitranscriptomics. Experiments investigating the mechanisms that underlie the downregulation of Notch1 expression in diabetic cardiac fibrosis revealed that Notch1 expression was decreased in HG/HF-induced CFs and mouse models of diabetic cardiac fibrosis likely due to aberrant Alkbh5 downregulation. 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 of Notch1 mRNA by YTHDF2, and induced Notch1 degradation. CF-specific Alkbh5 overexpression and Ythdf2 knockdown alleviated diabetic cardiac fibrosis by targeting Notch1. Our findings are consistent with those of previous studies showing that increased Notch1 expression in diabetic cardiac fibrosis inhibits the profibrotic phenotype associated with decreased m6A methylation and m6A-mediated phase separation.

Loss-of-function and gain-of-function approaches revealed that Notch1 downregulation promotes mitochondrial fission, which is required for CF proliferation. Mechanistically, Notch1-mediated transcriptional activation of Drp1 regulated mitochondrial fission. Notch1 deficiency inhibited the specific binding of Zeb1 to the Drp1 promoter thereby promoting Drp1 transcription. Hence, Notch1 overexpression prevented mitochondrial fission, proliferation, and collagen expression in CFs. Conversely, our data showed that Notch1 knockdown promoted mitochondrial fission and CF proliferation.

Although our study provides mechanistic insights, reduced Notch1 expression in diabetic hearts requires long-term therapeutic interventions as demonstrated in several animal models. Moreover, excessive mitochondrial fission likely affects many systemic cellular processes and signaling pathways not considered in this manuscript as we only focused on the mechanisms underlying CF proliferation and mitochondrial fission in diabetic cardiac fibrosis, and conditional gene knockouts and overexpression experiments were limited to mouse models. Neonatal CFs were chosen for the in vitro model due to their stability and accessibility. However, it is important to acknowledge the inherent differences in gene expression, structure, function, and response between fibroblasts from neonatal and adult mice. Although we showed that Alkbh5 and Ythdf2 regulate m6A methylation of Notch1 mRNA, m6A methylation has broader regulatory targets. Long-term interventions in ALKBH5−/− and YTHDF2−/− mice are required to explore the molecular mechanisms underlying diabetic cardiac fibrosis, which will be the subject of future research.

Data availability

No datasets were generated or analysed during the current study.

Abbreviations

AAV:

Adeno-associated virus

ALKBH5:

alkB homolog 5, RNA demethylase

CF:

Cardiac fibroblast

ChIP:

Chromatin immunoprecipitation

cTnT:

Cardiac troponin T

db/db:

Leptin receptor-deficient

DCM:

Diabetic cardiomyopathy

DRP1:

Dynamin-related protein 1

FIS1:

Mitochondrial fission 1

HFD:

High-fat diet

HG/HF:

High-glucose/high-fat

m6A:

N6-methyladenosine

mdivi-1:

Mitochondrial division inhibitor 1

MeRIP:

m6A-methylated RNA immunoprecipitation

METTL3:

Methyltransferase 3, N6-adenosine-methyltransferase complex catalytic subunit

MFF:

Mitochondrial fission factor

MID:

Mitochondrial division

NICD1:

NOTCH1 intracytoplasmic domain

NOTCH:

Notch homolog protein

Notch1-cKO:

Cardiac fibroblast-specific Notch1 conditional knockout

PCNA:

Proliferating cell nuclear antigen

STZ:

Streptozotocin

WTAP:

WT1 associating protein

FTO:

Fat mass and obesity-associated

YTHDF:

N6-methyladenosine RNA binding protein F

Zeb1:

Zinc finger E-box-binding homeobox 1

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Acknowledgements

The authors thank the ultrasound imaging system VINNO 6 (frequency 23 MHz, Vinno Corporation, Suzhou, China) for help with Echocardiography recordings.

Funding

This Study was supported by National Natural Science Foundation of China (82170236, 81700212), Key research and development projects of Anhui Province (202104j07020037), Excellent Youth Research Project in University of Anhui Province (2023AH030116), Research Fund of Anhui Institute of Translational Medicine (2021zhyx-C61), Excellent Top Talents Program of Anhui Province Universities (gxyqZD2022023) and National Natural Science Foundation Incubation Program of the Second Affiliated Hospital of Anhui Medical University (2020GMFY02), Anhui graduate education quality project (2023xscx053), Anhui Medical University graduate research and practice innovation project (YJS20230194).

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Author notes

  1. Zhi-Yan Liu, Li-Chan Lin, Zhen-Yu Liu and Kai Song have contributed equally to the first author

Authors and Affiliations

  1. Department of Anesthesiology and Perioperative Medicine, The Second Affiliated Hospital of Anhui Medical University, Hefei, 230601, People’s Republic of China

    Zhi-Yan Liu, Li-Chan Lin, Zhen-Yu Liu, Sui Mao, Ye Zhang, Rui Li & Hui Tao

  2. Department of Cardiothoracic Surgery, The Second Affiliated Hospital of Anhui Medical University, Hefei, 230601, People’s Republic of China

    Kai Song, Bin Tu, He Sun, Yang Zhou & Hui Tao

  3. Institute for Developmental and Regenerative Cardiovascular Medicine, MOE-Shanghai Key Laboratory of Children’s Environmental Health, Xinhua Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, 200092, People’s Republic of China

    Jian-Yuan Zhao

  4. Department of Clinical Pharmacology, The Second Affiliated Hospital of Anhui Medical University, Hefei, 230601, People’s Republic of China

    Jing-Jing Yang

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  1. Zhi-Yan Liu
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Contributions

LZY, LLC, and LZY were involved in the conception and study design and interpretation of the results. SK, TB, ZY, SH, MS were involved in the conduct of the study and interpretation of the results. ZY and LR were involved in the conception and the analysis of the results. YJJ, ZJY and TH wrote the first draft of the manuscript, All authors edited, reviewed, and approved the final version of the manuscript.

Corresponding authors

Correspondence to Rui Li, Jing-Jing Yang, Jian-Yuan Zhao or Hui Tao.

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Ethics approval and consent to participate

All animal procedures and experiments complied with NIH guidelines or Directive 2010/63/EU of the European Parliament on the Protection of Animals Used for Scientific Purposes and were approved by the ethics Committee of Anhui Medical University (The approval number is LLSC20232157). The clinical study for which written informed consent was obtained from all eligible patients, adhered to the principles of the Declaration of Helsinki; all experimental procedures involving human samples were reviewed and approved by the ethics committee of Anhui Medical University (The approval number is 2021061).

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Liu, ZY., Lin, LC., Liu, ZY. et al. N6-Methyladenosine-mediated phase separation suppresses NOTCH1 expression and promotes mitochondrial fission in diabetic cardiac fibrosis. Cardiovasc Diabetol 23, 347 (2024). https://doi.org/10.1186/s12933-024-02444-3

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Keywords

Cardiovascular Diabetology

ISSN: 1475-2840