Prepregnancy Obesity Reprograms Offspring Skeletal Muscle Fibre Transition Through H3K9me3

2.1 Animals

Four-week-old C57BL/6J mice were purchased from GemPharmatech Co. Ltd. (Jiangsu, China). Female mice were given either a standard diet or a high-fat diet (60%) before pregnancy, followed by a standard diet during pregnancy and lactation. Male mice used for breeding were continuously maintained on a chow diet. After 8 weeks of feeding, pregnancy was determined by the presence of a vaginal plug and assigned the embryonic age E0.5. All offspring were weaned onto a standard CD postnatally. Animals were anaesthetised with 1% pentobarbital before tissue collection. Samples were harvested at embryonic day 18.5 (E18.5) and at 8 weeks of age in offspring. Immediately after collection, tissues were flash-frozen in liquid nitrogen and stored at −80°C for further analysis. All animal procedures complied with the guidelines and regulations of the Chinese Laboratory Animal Guidelines and were approved by the Animal Care and Use Committee of Tongji Hospital (Approval No.: TJH-202209006).

2.2 Grip Strength Measurement and Treadmill Running

Grip strength was assessed in 8-week-old mice using a digital grip strength meter (BIOSEB, Vitrolles, France). To perform the test, the mice were placed on a grid with their bodies horizontal and their tails were gently pulled to determine the peak force, which was normalised by lean body mass. Each mouse was subjected to three trials at intervals greater than 15 min.

Mice were tested for exercise tolerance using a treadmill (Jeung Do Bio & Plant). To acclimatise the mice to the treadmill, they were run at 10 m/min for 5 min per day for 3 days. The mice were then tested by running on Day 4. The treadmill was run at 10 m/min for the first 5 min and then increased by 2 m/min every 2 min to 46 m/min until exhaustion. The running time of each mouse was recorded. Exhaustion was defined as the animal's inability to run on the treadmill for 10 s despite mechanical stimulation.

2.3 Additional Methods

Additional details regarding the methods and materials are provided in the Supporting Information.

2.4 Statistical Analysis

Data are presented as mean ± SEM. Statistical analyses and graphical presentations were performed using the GraphPad Prism 9.1 software. Normality and homogeneity of variance were tested before applying parametric tests. Two-group comparisons were conducted using an unpaired Student's t-test, while multiple group comparisons were performed using one-way ANOVA followed by Dunnett's post-hoc test. Significant differences are indicated as p < 0.05.

3.1 Maternal Prepregnancy Obesity Induces Systemic and Muscular Insulin Resistance in Offspring

After 8 weeks of HFD feeding (60% calorie from fat), the maternal mice developed obesity, evidenced by a greater than 20% increase in body weight. Additionally, they developed hyperlipidaemia, higher hepatic lipid content, as well as elevated fasting blood glucose and insulin levels (Supplementary Figure 1A–E). The litter sizes and survival rates of offspring from different groups were comparable (Supplementary Figure 2A,B).

In the offspring, both male and female of mothers with prepregnancy obesity (mHFD) had significantly increased birth weights compared with those of mothers with a prepregnancy chow diet (mCD). These weight differences persisted until weaning but declined in female offspring during subsequent growth (Figure 1B and Supplementary Figure 3A). At 8 weeks of age, although no weight differences were observed in females, fasting glucose and fasting insulin were significantly elevated in both male and female mHFD (Figure 1C and Supplementary Figure 3B,C). Consistently, mHFD exhibited significantly higher blood glucose levels at 30 min (19.27 ± 0.74 vs. 15.84 ± 0.89 mmol/L, p < 0.01) and 60 min (13.84 ± 1.15 vs. 11.61 ± 0.42 mmol/L, p = 0.087) during the glucose tolerance test (GTT) compared with mCD (Figure 1D). Furthermore, the area under the curve (AUC) for glucose tolerance was 12.87% higher in mHFD (p < 0.01) (Figure 1D). During the insulin tolerance test (ITT), mHFD exhibited a slower decline in blood glucose levels, with levels remaining significantly higher at 30 min (4.36 ± 0.09 vs. 3.69 ± 0.21 mmol/L, p < 0.01) and 120 min (6.79 ± 0.29 vs. 5.64 ± 0.14 mmol/L, p < 0.01) compared with mCD offspring (Figure 1E). Similar results were observed in female offspring (Supplementary Figure 3D,E). These results indicate that mHFD exhibited impaired glucose tolerance and insulin resistance.

Considering the pivotal role of skeletal muscle in glucose utilisation, we further investigated the insulin signalling pathway in muscle tissue of the offspring. The results indicated disrupted insulin signalling in mHFD offspring. Serine phosphorylation of IRS1 (Ser636) was significantly reduced to 26.33% (p < 0.0001) in the skeletal muscle of mHFD offspring compared with mCD offspring (Figure 1F). Similarly, AKT (Ser473) phosphorylation reduced by 50.4% (p < 0.01) (Figure 1F). A similar downward trend was also observed in female offspring (Supplementary Figure 3F). These findings suggested that maternal prepregnancy obesity leads to systemic and muscular insulin resistance in the offspring, with similar effects observed in both sexes.

3.2 Maternal Prepregnancy Obesity Reduces Oxidative Muscle Fibre in Offspring

To further investigate alterations in muscle tissue of offspring, we assessed the exercise capacity of both groups. The group of mHFD showed a greater muscle grip strength, shorter running duration and reduced time to exhaustion during running tests (Figure 2A and Supplementary Figure 5A). These findings indicated that mHFD performed better in explosive exercises but had diminished endurance during prolonged physical activity, suggesting a reduction of oxidative muscle fibres.

We observed that both male and female mice in the mHFD group had a higher tibialis anterior (TA) muscle weight, while no significant differences were found in gastrocnemius (GAS) and quadriceps (QU) muscle weights between the two groups (Supplementary Figure 4A,B). To better assess relative skeletal muscle mass changes, we calculated the ratio of muscle weight to body weight (Figure 2B and Supplementary Figure 5B). Further analysis revealed that the GAS ratio was significantly lower, whereas the TA ratio was significantly higher in both male and female mHFD mice. In contrast, the QU ratio showed no statistically significant difference between the groups.

Immunofluorescence staining showed that the average cross-sectional area of individual muscle fibres was significantly larger in TA (1885 ± 183.9 μm² vs. 1290 ± 149.3 μm², p < 0.05), QU (1828 ± 110.4 μm² vs. 1355 ± 99.75 μm², p < 0.01), GAS (2043 ± 119.5 μm² vs. 1655 ± 56.67 μm², p < 0.01) in the mHFD group compared with the mCD group (Figure 2C and Supplementary Figure 5C). Furthermore, the staining for muscle fibre types showed that mHFD possessed a lower proportion of Type I fibres (8.66 ± 1.32% vs. 22.97 ± 1.31%, p < 0.001) and a higher percentage of Type IIb fibres (91.32 ± 1.32% vs. 77.37 ± 1.38%, p < 0.001) in the GAS in comparison with mCD (Figure 2D and Supplementary Figure 5D). In mHFD, the protein expression level of the slow-twitch oxidative fibre marker Myh7 was significantly reduced. Concurrently, the expression of the fast-twitch glycolytic fibre marker Myh4 was notably higher, while the expression of the fast-twitch oxidative marker Myh2 remained unchanged (Figure 2E and Supplementary Figure 5E). As expected, the activity of the oxidative enzyme succinate dehydrogenase (SDH) was lower, while the activity of the glycolytic enzyme lactate dehydrogenase (LDH) was significantly higher in mHFD (Figure 2F and Supplementary Figure 5F). These results strongly suggested that maternal prepregnancy HFD exposure induced the transition of muscle fibre types, characterised by a reduction in slow-twitch oxidative fibres and an increase in fast-twitch glycolytic fibres.

3.3 Maternal Prepregnancy Obesity Alters IDH2 Expression in the Muscle Tissue of Offspring

To explore the underlying molecular mechanisms, we performed RNA sequencing (RNA-seq) analysis on the GAS from both mCD and mHFD. Principal component analysis (PCA) revealed that PC1 and PC2 accounted for 51.09% and 15.07% of the total gene expression variance, respectively (Figure 3A). A total of 2170 differentially expressed genes were identified (Figure 3B). GO analysis showed that these genes were involved in multiple signalling pathways associated with biological processes and cellular component, such as muscle system process, mitochondrion, transition between fast and slow fibre and glucose homeostasis (Figure 3C). KEGG pathway analysis further indicated associations with pathways including ECM-receptor interaction and PI3K-Akt signalling pathway (Figure 3D). Because the skeletal muscle fibre characteristics of offspring from prepregnancy obese dams were primarily marked by a significant reduction in the proportion of slow-twitch oxidative fibres, we focused our investigation on the screening and validation of downregulated genes. Because GO analysis revealed significant enrichment of mitochondrial function-related gene sets, we ranked differentially expressed genes based on their log fold change and selected 10 highly altered candidate genes for validation using PCR. The selection criteria included genes that were both significantly downregulated (adjusted p < 0.05) and had the highest absolute log fold change values. Among these genes, mitochondrial NADP-dependent isocitrate dehydrogenase 2 (IDH2) exhibited the most pronounced reduction in mRNA expression, showing a 29.67% lower expression in the mHFD group compared with the control mCD group. (Figure 3E). Additionally, we also observed significant differences in the protein levels of IDH2 between the two groups, with a 30.15% reduction in male offspring (p < 0.01) and a 46.02% reduction in female offspring (p < 0.0001), further supporting its potential role in the skeletal muscle of mHFD offspring (Figure 3F).

3.4 Muscle-Specific IDH2 Regulates Systemic Insulin Resistance in mHFD by Modulating Muscle Fibre Type Transition

To investigate the role of IDH2 in muscle fibre type transition, we performed an in vitro siRNA-mediated knockdown of IDH2 (si-IDH2) in the C2C12 skeletal muscle cell line. Remarkably, compared with cells transfected with the negative control (si-NC), si-IDH2 triggered the transition of muscle fibre types, with immunostaining evidence showing a significant reduction of the slow-twitch oxidative fibres and a corresponding elevation of the fast-twitch glycolytic fibres (Figure 4A). Further molecular studies revealed that the mRNA expression of Type I (oxidative slow-twitch) fibre-specific genes was significantly lower, including Myh7 (0.66-fold, p < 0.001), Tnni1 (0.74-fold, p < 0.001), Tnnc1(0.71-fold, p < 0.05), Myl3 (0.77-fold, p < 0.05) and Myoz2 (0.76-fold, p < 0.01). The expression of Type IIa fibre-specific genes, including Myh2 and Tnni2, showed no significant changes. In contrast, Type IIb fibre-specific genes were significantly increased, including Myh4 (1.68-fold, p < 0.001), Myh1 (1.54-fold, p < 0.001) and Pgm2 (1.4-fold, p < 0.001) (Figure 4B,C). These results underscored the critical regulatory role of IDH2 in muscle fibre type transition.

To further elucidate the role of IDH2 in muscle fibre transition and systemic metabolism in vivo, we utilised a tissue-specific IDH2 overexpression approach using lentiviral vectors. Specifically, at the age of 4 weeks, either a control lentivirus (LV-Control) or an IDH2 overexpressing lentivirus (LV-IDH2) was injected into the GAS, TA and QU muscles of offspring. Four weeks after injection, the overexpression of IDH2 was observed to reduce the cross-sectional area of muscle fibres in the GAS (Figure 5A). Notably, IDH2 overexpression partially restored the reduction of Type I fibres in mHFD, while simultaneously reducing the proportion of Type IIb fibres in the GAS (Figure 5A). Consistently, the overexpression of IDH2 significantly upregulated Myh7 expression (3.26-fold, p < 0.001) and suppressed Myh4 expression (0.39-fold, p < 0.0001) in mHFD (Figure 5B). More importantly, we found that muscle-specific IDH2 overexpression reduced fasting glucose levels (7.675 ± 0.177 mmol/L vs. 6.55 ± 0.157 mmol/L, p < 0.01) and fasting insulin levels (0.24 ± 0.01 ng/mL vs. 0.21 ± 0.005 ng/mL, p < 0.05) in mHFD offspring (Figure 5C,D) and alleviated impaired glucose tolerance and insulin sensitivity (Figure 5E,F). These data significantly implied that muscular IDH2 ameliorates systemic insulin sensitivity in offspring exposed to maternal prepregnancy obesity, by facilitating the transition of muscle fibres from fast-twitch glycolytic fibres to slow-twitch oxidative fibres.

3.5 IDH2 Promotes Mitochondrial Biogenesis Through the Maintenance of Redox Homeostasis

Given the strong link between mitochondrial biogenesis and muscle fibre composition, we hypothesised that IDH2 modulates muscle fibre transition by maintaining redox homeostasis and enhancing PGC-1α-mediated mitochondrial biogenesis in offspring exposed to maternal prepregnancy obesity.

To investigate mitochondrial abnormalities in mHFD offspring, we examined mitochondrial morphology, copy number and biogenesis-related gene expression. Electron microscopic analysis of GAS muscle revealed that mHFD exhibited mitochondrial swelling, disrupted cristae structure and reduced matrix density (Figure 6A). Compared with mCD, mitochondrial DNA (mtDNA) copy number was significantly reduced in mHFD offspring (0.69-fold, p < 0.01), suggesting impaired mitochondrial biogenesis (Supplementary Figure 6A). Furthermore, mRNA expression levels of key mitochondrial biogenesis markers were significantly lower in mHFD offspring, including PGC-1α (0.71-fold, p < 0.01), TFAM (0.79-fold, p < 0.05), TFB1m (0.77-fold, p < 0.05) and TFB2m (0.78-fold, p < 0.05), confirming impaired mitochondrial replication and transcription (Supplementary Figure 6B).

To further investigate the mechanisms by which IDH2 impacts mitochondrial function, we transfected C2C12 cells with si-IDH2. Flow cytometry revealed that compared with si-NC, C2C12 cells treated with si-IDH2 exhibited a significant increase in mitochondrial reactive oxygen species (ROS) levels (60.3 ± 2.65% in si-IDH2 vs. 32.0 ± 2.265% in si-NC, p < 0.01), coupled with a marked reduction in mitochondrial membrane potential, suggesting increased oxidative stress (Figure 6B,C). Consistently, we also observed that IDH2 knockdown led to an increase in the NADP⁺/NADPH ratio (1.62 ± 0.071 in si-IDH2 vs. 1.22 ± 0.121 in si-NC, p < 0.01) in cells (Supplementary Figure 6C). As expected, the expressions of the key mitochondrial biogenesis regulators, PGC1α and TFAM mRNA (Supplementary Figure 6D) and protein levels (Supplementary Figure 6E) were significantly decreased in si-IDH2-treated cells.

To confirm the role of IDH2 in mitochondrial biogenesis, we performed in vivo muscle-specific lentiviral overexpression of IDH2 in mHFD. IDH2 overexpression improved mitochondrial morphology, as evidenced by more compact cristae structure and enriched mitochondrial matrix density (Figure 6A). NADP+/NADPH levels were restored following IDH2 overexpression in skeletal muscle, confirming the role of IDH2 in maintaining redox homeostasis (Figure 6D). Consistently, IDH2 overexpression in mHFD significantly increased mitochondrial DNA content (Figure 6E) and restored the expression levels of PGC1α and TFAM (Figure 6F). These results suggest that IDH2 may promote mitochondrial biogenesis by regulating redox homeostasis, ultimately affecting fibre type composition in skeletal muscle.

To elucidate the underlying cause of reduced IDH2 levels in mHFD, we further analysed the expression levels of IDH2 and genes associated with mitochondrial biogenesis in foetal muscle. Compared with mCD, the mRNA expression of IDH2 was significantly lower in muscle tissue of mHFD (Supplementary Figure 6F). Consistently, mtDNA content (0.67-fold, p < 0.001) (Supplementary Figure 6G), as well as the expression levels of PGC1α (0.71-fold, p < 0.01), TFAM (0.72-fold, p < 0.05), TFB1m (0.75-fold, p < 0.05) and TFB2m (0.77-fold, p < 0.05) were reduced in the foetal muscle of mHFD (Supplementary Figure 6F). These results strongly implied an intra-uterine epigenetic modulation of IDH2 in offspring exposed to maternal prepregnancy obesity.

3.6 Prepregnancy Histone 3 Lysine 9 Trimethylation Modification in Oocyte Contributes to IDH2 Gene Downregulation in Muscle Tissue of mHFD

Histone modifications, particularly acetylation and methylation, are crucial for intra-uterine epigenetic control of protein expression. Acetylation of lysine 9 and lysine 27 on histone H3 (H3K9ac and H3K27ac) is associated with transcriptional activation, while tri-methylation of histone H3 on lysine 9, lysine 27 and lysine 36 (H3K9me3, H3K27me3 and H3K36me3) leads to transcriptional repression [25]. We conducted analysis of histone modifications in the skeletal muscle of mCD and mHFD. Compared with mCD, the mHFD exhibited a pronounced elevation in the levels of H3K9ac (1.78-fold, p < 0.05), H3K9me3 (2.41-fold, p < 0.0001) and H3K27ac (1.43-fold, p < 0.01), whereas the levels of H3K4me3, H3K36me3 and H3K36me3 remained unchanged (Figure 7A). Given that IDH2 expression was lower in mHFD mice, we focused on the investigation of H3K9me3, which is implicated in transcriptional silencing. ChIP-qPCR analysis revealed that H3K9me3 enrichment at the IDH2 promoter was higher in both male (2.54-fold, p < 0.0001) and female (2.55-fold, p < 0.0001) mHFD mice (Figure 7B).

Given that maternal environmental exposures can alter epigenetic profiles of gametes and early embryos, we hypothesised that the origin of H3K9me3 modification might trace back to the gamete stage. We analysed the expression levels of several histone methyltransferases (EHMT1, EHMT2, JMJD2A, JMJD2B, SETDB1, SETDB2, EZH2, ASH1L, JMJD1A, NSD2) and demethylases (KDM5A and PHF8) in the ovaries of CD and HFD dams. Among these, the expression level of the histone methyltransferases SETDB2 was significantly higher in HFD dams (Figure 7C,D). Moreover, the increased expression of SETDB2 was co-localised with the H3K9me3 histone modification in ovaries of HFD dams (Figure 7E). Collectively, our results suggested that reduced IDH2 expression in muscle tissue of mHFD may be attributed to H3K9me3 histone modification, which was likely initiated by the upregulated expression of SETDB2 in the oocytes of HFD dams.