Fasting Enhances Cardiomyocyte Hypoxia Tolerance by Regulating Ca²⁺ Transport at Mitochondria–Endoplasmic Reticulum Contact Sites

Abstract

Mitochondria–endoplasmic reticulum contacts (MERCs) are physical structures formed between mitochondria and the endoplasmic reticulum (ER) through various tethering proteins, playing crucial roles in multiple physiological processes, including Ca²⁺ and lipid exchange between the ER and mitochondria, regulation of mitochondrial morphology and dynamics (fusion and fission), as well as the induction of autophagy and apoptosis. Mitofusin 2 (MFN2), a key mitochondrial fusion protein, has been identified as an essential structural component of MERCs. Our research demonstrates that 16:8 circadian intermittent fasting (CIF) leads to enhanced mitochondrial fusion. The upregulation of MFN2 reinforces MERC stability, thereby facilitating efficient Ca²⁺ transfer between the ER and mitochondria. This process sustains the activity of mitochondrial oxidative phosphorylation (OXPHOS) enzymes, elevates mitochondrial oxygen utilization efficiency, and ultimately augments ATP production. Consequently, these adaptations enhance cardiomyocyte tolerance to hypoxic conditions. This study elucidates a novel mechanism by which MERCs regulate cellular hypoxia resistance and proposes a potential therapeutic strategy for improving acute hypoxia tolerance through the modulation of Ca²⁺ transport at MERCs.

1. Introduction

In recent years, the growing need for rapid deployment to high-altitude regions has underscored the urgency of developing effective strategies for swift adaptation to hypoxic conditions. Substantial evidence indicates that individuals ascending abruptly to altitudes above 2500 m without prior acclimatization are highly susceptible to acute hypoxia-related pathologies, including acute mountain sickness (AMS), high-altitude pulmonary edema (HAPE), and high-altitude cerebral edema (HACE), which can progress to life-threatening complications [1,2]. Currently, the most commonly utilized pre-acclimatization strategies include stepwise altitude acclimatization, intermittent hypoxia exposure, and pharmacological interventions. However, these approaches either require prolonged durations, making them impractical for rapid high-altitude access, or are associated with significant pharmacological side effects [3,4,5,6]. To date, no reliable method exists to achieve rapid hypoxic adaptation, and the underlying mechanisms for enhancing acute hypoxia tolerance remain poorly understood.

Our previous study demonstrated that three-day fasting significantly enhances the survival rate of rats exposed to hypoxic conditions equivalent to 7620 m altitude for 24 h. This protective effect is mediated through mTOR suppression, which attenuates metabolic synthesis while augmenting mitochondrial autophagy [7], suggesting that fasting represents a viable strategy for rapid acute hypoxic adaptation. However, prolonged fasting in humans leads to physical debilitation, thereby limiting its clinical applicability. To address this constraint, the present study employs a 16:8 CIF protocol in rats, which maintains baseline physical vigor while evaluating its effects on hypoxia tolerance.

MERCs are dynamic structures physically linked between the ER and mitochondria by tethering proteins. These contact sites play crucial roles in various fundamental cellular processes, including Ca²⁺ and lipid transport, mitochondrial ATP synthesis, and mitochondrial fusion and fission [8]. Intracellular Ca²⁺ is primarily stored in the ER and released into the cytosol upon stimulation. In non-excitable cells, the inositol 1,4,5-trisphosphate receptor (IP3R) serves as the ER Ca²⁺ release channel, while the voltage-dependent anion channel (VDAC) on the outer mitochondrial membrane is responsible for Ca²⁺ uptake. Both IP₃R and the isoform VDAC1 are enriched at MERC sites, where they interact via glucose-regulated protein 75 (Grp75) to form the VDAC-Grp75-IP3R complex. This complex mediates Ca²⁺ transfer from the ER to mitochondria, thereby enhancing OXPHOS and increasing ATP synthesis [9]. This process is regulated by the length, distance, and number of MERCs.

Mitochondria maintain the integrity of their morphological and functional capacity through the dynamic processes of fission and fusion, thereby enabling adaptation to metabolic changes or cellular stress. Studies have demonstrated that starvation conditions can promote mitochondrial fusion [10]. Mitofusin 2(MFN2), an outer mitochondrial membrane fusion protein, not only participates in mitochondrial fusion but is also recognized as a key tethering protein between mitochondria and the ER. In the present study, we found that 16:8 CIF improved the 24 h survival rate of rats exposed to an altitude of 7620 m. Mechanistically, 16:8 CIF upregulated MFN2 expression, which enhanced the structural stability of MERCs. This stabilization facilitated Ca²⁺ transfer from the ER to mitochondria, preserved the activity of mitochondrial OXPHOS enzymes, improved mitochondrial oxygen utilization efficiency, and ultimately maintained ATP production under hypoxic conditions.

2. Results

2.1. CIF Enhances Survival Rate, Reduces Myocardial Damage, and Preserves Cardiac Function in Rats After Acute Hypoxia Exposure

The survival rate of normal rats after 24 h of acute hypoxia exposure at 7620 m was 44.4%, while the survival rate of CIF-treated rats significantly increased to 83.3% (Table 1, p = 0.0354). To assess cardiac function of rats, we conducted echocardiographic examinations 5 h after acute hypoxic exposure (Figure 1A). The results showed that the left ventricular ejection fraction (LVEF) and left ventricular shortening fraction (LVSF) of rats significantly decreased after acute hypoxia (p < 0.01), and the heart rate showed an increasing trend. In contrast, there was no significant decrease in cardiac function in the CIF-treated rats (Figure 1B–D). Histological examination via HE staining of myocardial tissue revealed that rats with acute hypoxia exhibited disordered arrangement and rupture of myocardial fibers. Remarkably, no obvious myocardial injury was observed in the myocardial tissue of CIF-treated rats (Figure 1E,F). These findings collectively indicate that CIF confers significant cardioprotective effects against acute hypoxic stress.

Table 1.

Survival rates of rats after staying at an altitude of 7620 m for 24 h.

	Con	CIF
Total	18	18
Survival	8	15
Survival rate (%)	44.4	83.3

Con, control group (without fasting pretreatment); CIF, circadian intermittent fasting pretreatment group. Survival rate comparisons between groups were analyzed by a chi-square test.

Figure 1.

CIF alleviated myocardial injuries and maintained cardiac function under acute hypoxia. (A) Representative myocardial M-mode echocardiography images. Echocardiography was performed in the following stages: control under normoxic conditions without CIF treatment (N + Con), acute hypoxia exposure at 7620 m for 5 h (H + Con), circadian intermittent fasting under normoxic conditions (N + CIF), and CIF treatment combined with acute hypoxia (H + CIF). (B–D) Quantitative analysis of heart rate, left ventricular ejection fraction (LVEF), and left ventricular fraction shortening (LVFS). (E) Hematoxylin–eosin (HE) staining of myocardium, scale bar = 50 μm and 20 μm (enlarged diagram). (F) Pathology injury score of HE staining. Data are presented as mean ± SEM (n = 6 rats/group). ** p < 0.01 vs. the N + Con group; ^# p < 0.05 or ^## p < 0.01 vs. the H + Con group, analyzed by two-way ANOVA in (B–D,F).

2.2. CIF Attenuates Myocardial Apoptosis and ROS Production in Rats Exposed to Acute Hypoxia

Cardiomyocyte apoptosis was evaluated using TUNEL staining, and WGA was used to label cardiomyocytes (Figure 2A). The apoptosis rates did not differ significantly between N + Con (0.83 ± 0.14%) and N + CIF (0.75 ± 0.35%) groups. After acute hypoxia exposure, the myocardial apoptosis rate in the H + Con group reached 8.39 ± 0.10% (p < 0.01), showing a highly significant increase, whereas the myocardial apoptosis rate in the H + CIF group increased to 1.17 ± 0.15% and was significantly lower than that in the H + Con group (p < 0.01, Figure 2B).

Figure 2.

CIF reduced apoptotic rate and ROS production in myocardium. (A) Representative immunofluorescent images of the myocardium stained with wheat germ agglutinin (WGA, green), TUNEL (red), and DAPI (blue). Scale bar = 20 μm. (B) Quantitative analysis of the percentage of TUNEL-positive cells in the myocardium (n = 3–6 rats/group). (C,D) Representative Western blot bands (C) and quantitative analysis of cleaved/pro-Caspase 3 ratios in the myocardium (D); n = 3 rats/group). (E) Representative images of myocardium stained with dihydroethidium (DHE, red) and DAPI (blue). Scale bar = 50 μm. (F) Quantitative analysis of the mean fluorescence density of DHE in the myocardium (n = 3–6 rats/group). Data are presented as mean ± SEM. * p < 0.05 or ** p < 0.01 vs. the N + Con group; ^# p < 0.05 or ^## p < 0.01 vs. the H + Con group, analyzed by two-way ANOVA in (B,D,F).

Western blot analysis revealed that the ratio of cleaved caspase-3 to pro-caspase-3 in myocardium of rats in the N + CIF group showed no significant change compared with the N + Con group, but was significantly increased in the H + Con group (p < 0.05). In contrast, the H + CIF group showed no significant change compared with the N + Con group and was significantly lower than the H + Con group (p < 0.05, Figure 2C,D). Oxidative stress assessment using dihydroethidium (DHE) staining demonstrated that CIF significantly mitigated hypoxia-induced ROS production in rat myocardial cells (Figure 2E). Compared with the N + Con group, myocardial ROS content in the N + CIF group showed no obvious change. After acute hypoxia exposure, myocardial ROS content in the H + Con group increased significantly (p < 0.01), whereas that in the H + CIF group was significantly lower than that in the H + Con group (p < 0.01, Figure 2F).

2.3. CIF Enhances OXPHOS and Maintains ATP Production Under Acute Hypoxic Conditions

Cytochrome C oxidase subunit IV (COX IV) and citrate synthase (CS) are two critical enzymes involved in the mitochondrial OXPHOS process, both known to be calcium-dependent regulators of energy metabolism [8]. Western blot (WB) analysis revealed no significant differences in COX IV and CS protein expression among groups, with only marginal downward trends observed following hypoxia exposure (Figure 3A–C). The enzymatic activities of COX IV and CS were detected by enzyme activity assays (ELISA). The results showed that the activities of both COX IV and CS enzymes were increased after CIF (p < 0.01), decreased after hypoxia (p < 0.01), and also significantly reduced in the H + CIF group (p < 0.05, p < 0.01); however, they were markedly elevated compared with the H + Con group (p < 0.05, Figure 3D,E). Measurements of myocardial ATP content in rats showed that the ATP level was 10.94 ± 0.39 μmol/kg wet tissue in the N + Con group and 12.25 ± 0.54 μmol/kg wet tissue in the N + CIF group, with no significant difference observed between the two groups. After acute hypoxia exposure, the myocardial ATP content in the H + Con group significantly decreased to 9.37 ± 0.41 μmol/kg wet tissue (p < 0.05), whereas in the CIF + H group, it was 12.80 ± 0.66 μmol/kg wet tissue, significantly higher than that in the H + Con group (p < 0.05, Figure 3F).

Figure 3.

CIF enhanced OXPHOS-related enzyme activities and promoted ATP production in myocardial tissue. (A–C) Representative Western blot bands (A) and quantitative analysis of COX IV and CS protein levels in myocardial tissue (B,C); n = 3 rats/group). (D,E) Enzyme activity assay analysis of COX IV and CS enzyme activity in myocardial tissues (n = 3–6 rats/group). (F) Quantitative analysis of ATP content in myocardial tissues (n = 3–6 rats/group). All data are presented as mean ± SEM. * p < 0.05 or ** p < 0.01 vs. the N + Con group; ^# p < 0.05 vs. the H + Con group, analyzed by two-way ANOVA in (B–F).

2.4. CIF Promotes MERCs Stabilization and Mitochondrial Fusion in Myocardium Under Acute Hypoxic Conditions

As shown in Figure 4A–C, the length of MERCs in the myocardium of N + Con group was 298.1 ± 16.61 nm, which significantly increased to 406.3 ± 22.94 nm after CIF treatment (p < 0.01). After acute hypoxia, the length of MERCs in the H + Con group was 252.5 ± 16.65 nm, while it increased to 546.4 ± 22.04 nm in the H + CIF group, showing significant differences compared with both the N + Con group and the H + Con group (p < 0.01). The distance of MERCs in the myocardium of N + Con group was 27.85 ± 3.25 nm, which tended to decrease to 21.26 ± 4.86 nm after fasting. After acute hypoxia exposure, the distance increased to 28.64 ± 2.95 nm in the H + Con group but decreased significantly to 13.39 ± 1.00 nm in the H + CIF group (p < 0.01 compared with both the N + Con group and the Con + H group). These findings indicate that under hypoxic conditions, CIF strengthens the physical coupling between mitochondria and ER. Analysis of mitochondrial length revealed that the average mitochondrial length in the N + Con group was 1056 ± 46.51 nm, which elongated significantly to 1681 ± 82.50 nm after fasting (p < 0.01). After acute hypoxia exposure, the mitochondrial length of rats in the H + Con group was 926.9 ± 45.63 nm, while that in the H + CIF group was 1822 ± 109.70 nm, showing a significant increase compared with both the N + Con group and the H + Con group (p < 0.01, Figure 4D,E).

Figure 4.

CIF increased the length of MERCs, decreased the distance of MERCs and increased the expression of mitochondrial fusion-related proteins. (A) Representative transmission electron microscopy (TEM) images of myocardial tissue. Scale bar = 1 μm (70 k) and 500 nm (210 k). Red lines indicate the outline of MERCs, and yellow arrows indicate the distance between the ER and mitochondria. (B,C) Quantitative analysis of the length (B) and distance (C) of MERCs. Data were collected from 3 rats per group, with >3 fields of view measured per rat section. The full length of each MERC was measured, and the distance was averaged from 3 measurements at each MERC. (D,E) Representative images of myocardial electron microscopy (D); Scale bar = 2 μm (28 k) and the length statistics of mitochondria (E); n = 3 rats/group, 3 sections/rat). (F–K) Representative Western blot bands (F) and quantitative analysis of mitochondria fission and fusion-related proteins (FIS1, p-DRP1/DRP1; OPA1, MFN1, MFN2) in myocardial tissue (G–K); n = 3 rats/group). Data are presented as mean ± SEM. * p < 0.05 or ** p < 0.01 vs. the N + Con group; ^# p < 0.05 or ^## p < 0.01 vs. the H + Con group, analyzed by two-way ANOVA in (B,C,E,G–K).

To further investigate the underlying mechanisms, we assessed the expression of mitochondrial dynamics-related proteins, including mitochondrial fission-related proteins (FIS1, p-DRP1, DRP1) and fusion-related proteins (OPA1, MFN1, MFN2) in myocardial tissues. The results showed that OPA1 exhibited two bands at approximately 86 kDa and 92 kDa (Figure 4F). The expression of OPA1 was significantly increased in the N + CIF group (p < 0.01) and remained unchanged in the H + Con group, while in the H + CIF group, it was significantly higher than that in both the N + Con group and the H + Con group (p < 0.05, Figure 4G). The expression of MFN1 showed no significant change after either fasting or hypoxia treatment (Figure 4H). MFN2 levels increased significantly after fasting (p < 0.01). After hypoxia exposure, the MFN2 expression in the H + Con group showed no significant change, whereas it was significantly upregulated in the H + CIF group compared with both the N + Con group and the H + Con group (p < 0.05, Figure 4I). In contrast, the expression of fission-related proteins FIS1 and p-DRP1/DRP1 remained unaltered by either fasting or hypoxia exposure (Figure 4J,K), demonstrating the specific effects of CIF treatment on mitochondrial fusion pathways under hypoxia conditions.

2.5. Simulated CIF Reduced Apoptosis and Mitochondrial ROS Production in Cultured Rat Cardiomyocytes Under Acute Hypoxic Conditions

H9C2 rat cardiomyocytes were cultured in vitro and subjected to simulated CIF using low-glucose medium. As shown in Figure 5A,B, flow cytometry analysis demonstrated that the apoptosis rates of the N + Con and N + CIF group were 3.22 ± 0.06% and 3.73 ± 0.18%, respectively. After acute hypoxia exposure (1% O₂ for 24 h), the H + Con group exhibited a significant increase in apoptosis to 5.29 ± 0.10% (p < 0.01), whereas the H + CIF group exhibited a non-significant change (3.42 ± 0.13%) compared with the N + Con group and a significantly lower apoptosis rate compared with the H + Con group (p < 0.01). Mitochondrial ROS levels in cardiomyocytes were assessed using flow cytometry, and the results showed that there was no significant difference between the N + CIF and N + Con groups under normoxic conditions. After hypoxia, the H + Con group displayed a marked increase in mitochondrial ROS (p < 0.01). The ROS level in the H + CIF group showed no significant difference compared with the N + Con and H + Con groups, but a decreasing trend was observed relative to the Con + H group (Figure 5C,D). Western blot was used to detect the expression levels of cleaved/pro-caspase3. The results showed that there was no significant change in the expression of cleaved/pro-caspase3 after fasting, while it increased significantly after hypoxia (p < 0.05). Moreover, the ratio in the H + CIF group was significantly decreased compared with that in the H + Con group (p < 0.01, Figure 5E,F).

Figure 5.

Simulated CIF reduced apoptotic rate and mitochondrial ROS production in cardiomyocytes. (A,B) Flow cytometric analysis of apoptosis by Annexin V and PI double-staining in cardiomyocytes (A) and quantitative analysis of the proportion of apoptotic cells (B). (C,D) Flow cytometric analysis of mitochondrial ROS by MitoSOX staining in cardiomyocytes (C) and quantitative analysis of the mean fluorescence intensity (D). (E,F) Representative Western blot bands (E) and quantitative analysis of cleaved/pro-caspase 3 expression (F). Data are presented as mean ± SEM (n = 3). * p < 0.05 or ** p < 0.01 vs. the N + Con group; ^## p < 0.01 vs. the H + Con group, analyzed by two-way ANOVA in (B,D,F).

2.6. Simulated CIF Enhances OXPHOS-Related Enzyme Activities, Thereby Improving the Oxygen Utilization Efficiency and ATP Production in Cardiomyocytes

In vitro analysis of COX IV and CS protein expression levels revealed no significant changes following CIF or hypoxic treatment (Figure 6A–C). However, enzyme activity assay results demonstrated that the enzymatic activities of both COX IV and CS were significantly elevated after fasting (p < 0.01) and decreased after hypoxic exposure (p < 0.01). Notably, these two enzymes in the H + CIF group also showed significantly lower activities compared with the N + Con group, but markedly higher activities relative to the H + Con group (p < 0.01, Figure 6D,E).

Figure 6.

Simulated CIF enhances OXPHOS-related enzyme activities, and promoted oxygen utilization and ATP production in cardiomyocytes. (A–C) Representative Western blot bands (A) and quantitative analysis of COX IV and CS protein levels in cardiomyocytes (B,C). (D,E) Quantitative analysis of COX IV and CS enzyme activity in cardiomyocytes by enzyme activity assay. (F,G) Oxygen consumption rate (OCR) curves and statistical analysis of OXPHOS parameters (basal respiration, ATP production, maximal respiration) in cardiomyocytes. (H,I) Extracellular acidification rate (ECAR) curves and statistical analysis of glycolytic parameters (glycolysis, glycolytic capacity, glycolytic reserve) in cardiomyocytes. (J) Quantitative analysis of ATP content in cardiomyocytes. Data are presented as mean ± SEM (n = 3). * p < 0.05 or ** p < 0.01 vs. the N + Con group; ^# p < 0.05 or ^## p < 0.01 vs. the H + Con group, analyzed by two-way ANOVA in (B–E,G,I,J).

Mitochondrial respiratory function was assessed using the Seahorse XF Analyzer. Oxygen consumption rate (OCR) was measured to sequentially assess key functional parameters: basal respiration was first recorded, followed by the addition of oligomycin to inhibit ATP synthase, which allowed the quantification of ATP-linked respiration and proton leak. Subsequent uncoupling with FCCP induced maximal respiration, revealing the spare respiratory capacity. Finally, inhibition of mitochondrial complexes I and III with rotenone and antimycin A permitted the measurement of non-mitochondrial respiration. These results revealed that the basal respiration, ATP production efficiency, and maximal respiratory capacity remained unchanged after fasting. After acute hypoxia exposure, the H + Con group displayed significant reductions in these parameters (p < 0.01), whereas the H + CIF group showed significant recovery compared with the H + Con group (^# p < 0.05, ^## p < 0.01, Figure 6F,G).

The extracellular acidification rate (ECAR) assay was performed in glucose-free medium. Glucose was first added to provide substrate for glycolysis, allowing measurement of the basal glycolytic rate. Subsequently, oligomycin was introduced to inhibit mitochondrial oxidative phosphorylation, thereby forcing cells to rely on glycolysis for energy production and inducing the maximal glycolytic capacity. Finally, 2-deoxy-D-glucose (2-DG) was added to competitively block glycolytic flux, establishing the non-glycolytic acidification baseline for data normalization. The glycolytic reserve was then calculated as the difference between the maximal glycolytic capacity and the basal glycolytic rate. Based on these measurements, results indicated no significant changes in basal glycolysis, glycolytic capacity, or glycolytic reserve after fasting. However, acute hypoxia exposure significantly increased these glycolytic parameters in the H + Con group (* p < 0.05, ** p < 0.01), which were notably attenuated in the H + CIF group (* p < 0.05, ** p < 0.01, Figure 6H,I). These findings suggest that simulated CIF treatment promotes a metabolic shift toward OXPHOS rather than glycolysis under acute hypoxic conditions. Cellular ATP content measurements revealed that the ATP baseline levels were 0.24 ± 0.01 μmol/10⁹ cells in the N + Con group and 0.21 ± 0.01 μmol/10⁹ cells in the N + CIF group. After acute hypoxia exposure, ATP content decreased significantly to 0.18 ± 0.01 μmol/10⁹ cells in the H + Con group (p < 0.01), whereas the H + CIF group maintained ATP levels at 0.23 ± 0.01 μmol/10⁹ cells, significantly higher than the H + Con group (p < 0.05, Figure 6J).

2.7. Simulated CIF Promotes MERC Formation and Mitochondrial Fusion in Cultured Rat Cardiomyocytes Under Acute Hypoxic Conditions

The length and distance of MERCs in H9C2 cardiomyocytes were detected by transmission electron microscopy (Figure 7A). The results showed that the average length of MERCs in the N + Con group was 208.1 ± 27.76 nm, which significantly increased to 355.3 ± 25.60 nm following CIF treatment. After acute hypoxia exposure, the length of MERCs in the H + Con group was 244.6 ± 33.86 nm, showing no significant change compared with the N + Con group. Whereas the H + CIF group exhibited a marked increase to 631.6 ± 44.64 nm, demonstrating a significant increase compared with both the N + Con and H + Con groups (p < 0.01, Figure 7B). MERC distances in the N + Con and N + CIF groups were 27.02 ± 3.00 nm and 24.40 ± 1.92 nm, respectively. After acute hypoxia exposure, the distance of MERCs in the H + Con group was 32.45 ± 1.89 nm, with no significant change observed compared with the N + Con group. In contrast, the H + CIF group displayed a significant reduction to 16.32 ± 1.28 nm, which was notably shorter than those in both the N + Con and H + Con groups (p < 0.01, Figure 7C). Statistical analysis of mitochondrial length in cardiomyocytes revealed that the average mitochondrial length in the N + Con group was 572.5 ± 67.66 nm, which significantly increased to 859.0 ± 108.10 nm after fasting (p < 0.05). After hypoxia exposure, the mitochondrial length in the H + Con group was 593.0 ± 42.44 nm, showing no significant change compared with the N + Con group. However, in the H + CIF group, the mitochondrial length increased to 1176 ± 182.50 nm, which was significantly longer compared with both the N + Con and H + Con groups (p < 0.01, Figure 7D,E).

Figure 7.

Simulated CIF increased the length of MERCs, decreased the distance of MERCs and increased the expression of mitochondrial fusion-related proteins. (A) Representative electron microscopy images of cardiomyocytes. Scale bar = 200 nm. Red lines indicate the outline of MERCs, and yellow arrows indicate the distance between the ER and mitochondria. (B,C) Quantitative analysis of the length (B) and distance (C) of MERCs. n = 9 images from three independent experiments. The full length of each MERC was measured, and the distance was averaged from 3 measurements at each MERC. (D,E) Representative electron microscopy images of cardiomyocytes (D); scale bar = 200 nm) and the length statistics of mitochondria (E); n = 9 images from three independent experiments). (F–K) Representative Western blot bands (F) and quantitative analysis of mitochondria fission and fusion-related proteins (FIS1, p-DRP1/DRP1; OPA1, MFN1, MFN2) in cardiomyocytes (G–K); n = 3). Data are presented as mean ± SEM. * p < 0.05 or ** p < 0.01 vs. the N + Con group; ^# p < 0.05 or ^## p < 0.01 vs. the H + Con group, analyzed by two-way ANOVA in (B,C,E,G–K).

Western blot analysis of mitochondrial dynamics-related proteins (fusion: OPA1, MFN1, MFN2; fission: FIS1, p-DRP1, DRP1) showed consistent trends with those observed in rat myocardium (Figure 7F). After fasting, OPA1 expression was significantly upregulated in the N + CIF group (p < 0.05). After acute hypoxia exposure, no significant changes in OPA1 expression were observed in the H + Con group, whereas the H + CIF group showed a significant increase in OPA1 expression compared with both the N + Con and H + Con groups (p < 0.05, Figure 7G). MFN1 expression showed no significant changes after fasting or hypoxia treatment (Figure 7H). MFN2 expression increased significantly in the N + CIF group (p < 0.05) and decreased in the H + Con group (p < 0.05), whereas the H + CIF group displayed a significant increase compared with the H + Con group (p < 0.05, Figure 7I). Fission-related proteins (FIS1 and p-DRP1/DRP1) showed no significant changes across all groups (Figure 7J,K).

2.8. Overexpression of the MERC Structural Protein MFN2 Reduced Apoptosis and Mitochondrial ROS Production in Cultured Rat Cardiomyocytes Under Acute Hypoxic Conditions

MFN2 is an essential tethering protein at MERCs. Given that CIF induces pronounced alterations in MERC architecture and mitochondrial morphology, along with elevated MFN2 expression, we sought to investigate the functional role of MFN2 in mediating these adaptive responses. Since the VAPB-PTPIP51 complex is a well-characterized tether critical for MERC integrity [11], we first examined whether MFN2 colocalizes with PTPIP51, a core component of this complex, in H9C2 cells (Figure 8A). The results demonstrated clear colocalization between MFN2 and PTPIP51. Notably, this colocalization was significantly increased in the N + CIF group after fasting (p < 0.05), while hypoxia treatment induced no significant change. Importantly, the H + CIF group exhibited a pronounced elevation in colocalization compared with both the N + Con and H + Con groups (p < 0.01, Figure 8B). Subsequently, MFN2 was overexpressed in H9C2 cardiomyocytes using an adenoviral vector, with a multiplicity of infection (MOI) of 100 selected as the optimal viral infection condition (Figure 8C,D). Further detection of the colocalization between MFN2 and PTPIP51 was performed in an MFN2 overexpression model (Figure 8E). The results similarly revealed a significant colocalization between MFN2 and PTPIP51, and this colocalization was significantly enhanced following MFN2 overexpression (p < 0.01). Following hypoxic treatment, the colocalization of MFN2 and PTPIP51 was significantly reduced (p < 0.01), whereas the H + OE group exhibited a substantial increase in colocalization compared with both the N + Con group and the H + Con group (* p < 0.05, ^## p < 0.01, Figure 8F). The above results indicate that CIF can lead to increased expression of MFN2 localized at MERC structures.

Figure 8.

Overexpression of the MERC structural protein MFN2 reduced apoptotic rate and mitochondrial ROS production in cardiomyocytes. (A) Representative immunofluorescence images showing MFN2 (green) and PTPIP51 (red) in CIF-treated cardiomyocytes. Nuclei were counterstained with DAPI (blue). Yellow signal in the merge panel indicates colocalization of MFN2 and PTPIP51. Scale bar = 10 μm. (B) Quantitative colocalization analysis (Pearson correlation coefficient) between MFN2 and PTPIP51. Data are from three independent experiments; three fields of view were analyzed per replicate. (C,D) Representative Western blot bands (C) and quantitative analysis of MFN2 protein levels in H9C2 cells (D). The different multiplicities of infection (MOI) indicate varying viral titers used for adenovirus transduction. H9C2 cells were transduced with adenovirus encoding MFN2 at the indicated MOI, or with an empty vector control virus (Con). (E) Representative immunofluorescence images of MFN2 (green) and PTPIP51 (red) in MFN2 overexpression cardiomyocytes. Nuclei were counterstained with DAPI (blue). Yellow signal in the merge panel indicates colocalization of MFN2 and PTPIP51. Scale bar = 10 μm. Con: cells transduced with empty vector adenovirus. OE: cells transduced with adenovirus encoding MFN2 at an MOI of 100. (F) Quantitative colocalization analysis between MFN2 and PTPIP51. Data are from three independent experiments; three fields of view were analyzed per replicate. (G,H) Flow cytometric analysis of apoptotic rate by Annexin V and PI double-staining in H9C2 cells (G) and quantitative analysis of the proportion of apoptotic cells (H). (I,J) Flow cytometric analysis of mitochondrial ROS by MitoSOX staining in cardiomyocytes (I) and quantitative analysis of the mean fluorescence intensity (J). (K,L) Representative Western blot bands (K) and quantitative analysis of cleaved/pro-caspase 3 expression (L). Data are presented as mean ± SEM (n = 3). * p < 0.05 or ** p < 0.01 group vs. the N + Con group; ^## p < 0.01 vs. the H + Con group, analyzed using Student’s t-test in (D) and analyzed by two-way ANOVA in (B,F,H,J,L).

Next, we detected cell apoptosis via flow cytometry in the MFN2 overexpression model (Figure 8G). The results showed that the apoptosis rates in the control-vector infection (N + Con) group and the MFN2-overexpressing (N + OE) group were 5.70 ± 0.37% and 3.19 ± 0.18%, respectively. After exposure to 1% O₂ hypoxic conditions, the apoptosis rate increased to 7.71 ± 0.65% in the hypoxia plus control-vector infection (H + Con) group, whereas the hypoxia plus MFN2-overexpressing (H + OE) group exhibited a significant reduction to 3.64 ± 0.20% compared with both the N + Con and H + Con group (* p < 0.05, ^## p < 0.01, Figure 8H). Mitochondrial ROS levels, assessed by flow cytometry, were significantly decreased following MFN2 overexpression (p < 0.01). Acute hypoxic exposure induced a marked increase in mitochondrial ROS in the H + Con group (p < 0.01), while in the H + OE group, mitochondrial ROS levels were significantly reduced compared with the H + Con group (p < 0.01, Figure 8I,J). Western blot analysis of cleaved/pro-caspase-3 ratios showed no significant changes following MFN2 overexpression. After acute hypoxia, cleaved/pro-caspase-3 expression was upregulated in the H + Con group (p < 0.05), whereas MFN2 overexpression significantly attenuated this hypoxic-induced upregulation in the H + OE group (p < 0.01 vs. H + Con, Figure 8K,L).

2.9. Overexpression of MFN2 Enhances OXPHOS-Related Enzyme Activities, Thereby Improving the Oxygen Utilization Efficiency and ATP Production of Cardiomyocytes in Cultured Rat Cardiomyocytes

The protein expression levels of COX IV and CS were detected in vitro, and the results showed no significant changes following MFN2 overexpression or hypoxic treatment (Figure 9A–C). Enzyme activity assay results demonstrated that overexpression of MFN2 significantly increased the activities of COX IV and CS (p < 0.01). After hypoxic exposure, the activities of these two enzymes were markedly decreased (p < 0.01). Although the enzyme activities in the H + OE group were also significantly lower than those in the N + Con group (p < 0.01), they were notably elevated relative to the H + Con group (p < 0.05, Figure 9D,E). Mitochondrial respiratory function was assessed using a Seahorse XF Analyzer; the OCR results indicated that MFN2 overexpression did not alter basal respiration, ATP production efficiency, or maximal respiratory capacity under normoxic conditions. After acute hypoxia exposure, the H + Con group displayed significant reductions in basal respiration, maximal respiratory capacity and ATP production efficiency (p < 0.01). In contrast, the H + OE group showed a significant recovery in basal respiration (p < 0.01), maximal respiratory capacity (p < 0.01) and ATP production efficiency (p < 0.05) compared with the H + Con group (Figure 9F,G). The ECAR results indicated that MFN2 overexpression had no significant effect on basal glycolytic rate, glycolytic capacity, or glycolytic reserve of mitochondria. After acute hypoxia exposure, these glycolytic parameters increased significantly in the H + Con group (p < 0.01), whereas the H + OE group showed significant reductions in these indicators compared with the H + Con group (^# p < 0.05, ^## p < 0.01, Figure 9H,I). These findings suggest that MFN2 overexpression shifts mitochondrial energy metabolism toward OXPHOS rather than glycolysis under acute hypoxic conditions. Measurement of cellular ATP content revealed that the ATP levels in H9C2 cardiomyocytes were 0.13 ± 0.01 μmol/10⁹ cells in the N + Con group and 0.14 ± 0.02 μmol/10⁹ cells in the N + OE group. After acute hypoxia exposure, the ATP content in the H + Con group decreased significantly to 0.07 ± 0.01 μmol/10⁹ cells (p < 0.01), while that in the H + OE group decreased to 0.09 ± 0.01 μmol/10⁹ cells (p < 0.05); the latter, however, was significantly higher than that of the H + Con group (p < 0.05, Figure 9J).

Figure 9.

Overexpression of MFN2 enhanced OXPHOS-related enzyme activities, and promoted oxygen utilization and ATP production in cardiomyocytes. (A–C) Representative Western blot bands (A) and quantitative analysis of COX IV and CS protein levels in cardiomyocytes (B,C). (D,E) Quantitative analysis of COX IV and CS enzyme activity in cardiomyocytes by enzyme activity assay. (F,G) OCR curves and statistical analysis of OXPHOS parameters (basal respiration, ATP production, maximal respiration) in cardiomyocytes. (H,I) ECAR curves and statistical analysis of glycolytic parameters (glycolysis, glycolytic capacity, glycolytic reserve) in cardiomyocytes. (J) Quantitative analysis of ATP content in cardiomyocytes. Data are presented as mean ± SEM (n = 3). * p < 0.05 or ** p < 0.01 group vs. the N + Con group; ^# p < 0.05 or ^## p < 0.01 vs. the H + Con group, analyzed by two-way ANOVA in (B–E,G,I,J).

2.10. Overexpression of MFN2 Promotes MERC Formation and Mitochondrial Fusion in Cultured Rat Cardiomyocytes Under Acute Hypoxic Conditions

To assess the role of MFN2 in MERCs and mitochondrial dynamics, we analyzed H9C2 cardiomyocytes using transmission electron microscopy (Figure 10A). Quantitative analysis revealed that MERCs length in the N + Con group was 275.3 ± 24.46 nm, which significantly increased to 623.5 ± 64.74 nm following MFN2 overexpression (N + OE group, p < 0.05). After acute hypoxia exposure, MERCs length in the H + Con group (275.9 ± 28.38 nm) remained unchanged compared with the N + Con group, whereas the H + OE group displayed a pronounced increase to 1266 ± 168.90 nm, which was significantly longer than both the N + Con and H + Con groups (p < 0.01, Figure 10B). Similarly, the distance of MERCs in the N + Con group was 32.82 ± 3.59 nm. which was significantly reduced to 19.25 ± 1.47 nm in the N + OE group (p < 0.01). After acute hypoxia exposure, MERCs distance in the H + Con group (32.75 ± 3.77 nm) showed no significant change compared with the N + Con group, but in the H + OE group, it decreased to 19.90 ± 1.37 nm, which was significantly shorter than both the N + Con and H + Con groups (p < 0.01, Figure 10C). Mitochondrial morphology analysis demonstrated that MFN2 overexpression increased mitochondrial length from 684.5 ± 80.51 nm (N + Con) to 957.3 ± 77.58 nm (p < 0.05). After acute hypoxia exposure, the mitochondrial length in the H + Con group was 790.6 ± 103.30 nm, while in the H + OE group, it increased to 1175 ± 100.70 nm, showing a significant increase compared with both the N + Con and H + Con groups (** p < 0.01, ^# p < 0.05, Figure 10D,E). Western blot analysis was performed to detect the expression of mitochondrial dynamics proteins in H9C2 cardiomyocytes (Figure 10F). The results revealed that MFN2 overexpression (N + OE group) significantly upregulated OPA1 (p < 0.05). After acute hypoxia exposure, there was no significant change in the H + Con group, whereas the H + OE group showed a significant increase in OPA1 expression compared with the Con + H group (p < 0.05, Figure 10G). MFN2 expression was elevated in both OE and OE + H groups (p < 0.05), whereas MFN1 remained unchanged (Figure 10H,I). No significant alterations were observed in fission-related proteins (FIS1, p-DRP1/DRP1) after fasting or hypoxia treatment (Figure 10J,K).

Figure 10.

Overexpression of MFN2 increased the length of MERCs, decreased the distance of MERCs and increased the expression of mitochondrial fusion-related proteins in cardiomyocytes. (A) Representative electron microscopy images of cardiomyocytes. Scale bar = 200 nm. Red lines indicate the outline of MERCs, and yellow arrows indicate the distance between the ER and mitochondria. (B,C) Quantitative analysis of the length (B) and distance (C) of MERCs. n = 9 images from three independent experiments. The full length of each MERC was measured, and the distance was averaged from 3 measurements at each MERC. (D,E) Representative electron microscopy images of cardiomyocytes (D); scale bar = 200 nm) and the length statistics of mitochondria (E); n = 9 images from three independent experiments). (F–K) Representative Western blot bands (F) and quantitative analysis of mitochondria fission and fusion-related proteins (FIS1, p-DRP1/DRP1; OPA1, MFN1, MFN2) in cardiomyocytes (G–K); n = 3). Data are presented as mean ± SEM. * p < 0.05 or ** p < 0.01 vs. the N + Con group; ^# p < 0.05 or ^## p < 0.01 vs. the H + Con group, analyzed by two-way ANOVA in (B,C,E,G–K).

2.11. CIF Promotes MERC Formation and Mitochondrial-ER Ca²⁺ Transfer via MFN2 in Cultured Rat Cardiomyocytes

Mitochondria and the ER were labeled with Mitotracker (red) and ER-Tracker (green), respectively. The overlap of these fluorophores (yellow fluorescence) indicated close proximity between mitochondria and the ER (i.e., MERCs). Our results showed that CIF significantly increased the number of MERCs (p < 0.01). Following acute hypoxia exposure, the number of MERCs remained unchanged in the H + Con group, whereas a significant increase was observed in the H + CIF group (p < 0.01; Figure 11A,C). To assess the role of MFN2 in MERCs regulation, we further quantified MERCs in H9C2 cells with MFN2 overexpression. The results showed that MFN2 overexpression significantly increased the number of MERCs (p < 0.01). After acute hypoxia, the number of MERCs did not change significantly in the H + Con group, but was markedly elevated in the H + OE group (p < 0.01; Figure 11B–D).

Figure 11.

Simulated CIF increased the number of MERCs and promoted Ca²⁺ transport between the ER and mitochondria via MFN2. (A) Representative fluorescence images depicting MERCs in cardiomyocytes from CIF groups. Mitochondria were labeled with MitoTracker (red), the endoplasmic reticulum (ER) with ER-Tracker (green), and nuclei with hoechst33342 (blue). Yellow regions in the merged image indicate apposition of red mitochondrial and green ER signals, representing putative MERCs. ZOOM shows a higher magnification view of the boxed area. Scale bar = 20 μm. (B) Representative images of the number of MERCs in cardiomyocytes in MFN2-OE groups. Scale bar = 20 μm. (C,D) Pearson correlation coefficient of mitochondria and the ER in CIF groups (C) and MFN2-OE groups (D). ** p < 0.01 vs. the N + Con group; ^## p < 0.01 vs. the H + Con group. (E,F) Representative fluorescence images of cardiomyocytes (E) and quantification of Rhod-2 fluorescence intensity (F) in the CIF-treated and CIF + 2APB-treated groups. Mitochondria were labeled with MitoTracker (green) and mitochondrial Ca²⁺ with Rhod-2 probe (red), with merged images indicating colocalization. ZOOM represents a higher magnification view of the boxed area. Scale bar = 20 μm. (G,H) Representative images (G) and quantification of mitochondrial Ca²⁺ levels (H) in the OE and OE + 2APB-treated groups. Scale bar = 20 μm. (I,J) Representative Western blot bands (I) and quantitative analysis of IP3R protein levels (J) in H9C2 cells transfected with si-IP3R or si-NC (negative control). (K,L) Representative fluorescence images of cardiomyocytes (K) and quantification of Rhod-2 fluorescence intensity (L) in the CIF-treated and CIF + si-IP3R treated groups. Mitochondria were labeled with MitoTracker (green) and mitochondrial Ca²⁺ with Rhod-2 probe (red), with merged images indicating colocalization. ZOOM represents a higher magnification view of the boxed area. Scale bar = 20 μm. (M,N) Representative images (M) and quantification of mitochondrial Ca²⁺ levels (N) in the OE and OE + si-IP3R treated groups. Scale bar = 20 μm. Data are presented as mean ± SEM (n = 3). * p < 0.05 or ** p < 0.01, analyzed by two-way ANOVA in (C,D,F,H,J,L,N).

Furthermore, Rhod-2 (red) was used to label mitochondrial Ca²⁺, and mitochondria were co-labeled with Mitotracker (green). The IP3R inhibitor 2-APB was used to block ER-derived Ca²⁺ transport (Figure 11E). Our results showed that CIF significantly increased mitochondrial Ca²⁺ levels (p < 0.01). Following acute hypoxia, mitochondrial Ca²⁺ remained unchanged in the H + Con group, whereas the H + CIF group exhibited a significant increase compared with the H + Con group (p < 0.05), indicating that CIF promotes mitochondrial Ca²⁺ influx. Notably, 2APB treatment led to a significant reduction in mitochondrial Ca²⁺ in the N + Con + 2-APB, N + CIF + 2-APB, and H + CIF + 2-APB groups compared with their respective untreated controls. A decreasing trend was also observed in the H + Con + 2-APB group relative to H + Con, though this did not reach statistical significance (Figure 11F). These findings suggest that CIF-induced mitochondrial Ca²⁺ elevation is derived from the ER. Consistent results were obtained in the MFN2 overexpression model (Figure 11G). MFN2 overexpression significantly increased mitochondrial Ca²⁺ (p < 0.05); Following acute hypoxia, mitochondrial Ca²⁺ remained unchanged in the H + Con group, but was significantly higher in the H + OE group compared with H + Con (p < 0.01). Consistently, 2-APB treatment reduced mitochondrial Ca²⁺ in the N + Con + 2-APB, N + OE + 2-APB, and H + OE + 2-APB groups relative to their untreated controls, with a non-significant trend in H + Con + 2-APB (Figure 11H). To further validate these findings, IP3R expression was silenced using small interfering RNA (siRNA). We verified the knockdown efficiency by Western blot (Figure 11I,J). Subsequently, mitochondrial Ca²⁺ levels were measured in the fasting cell model (Figure 11K). The results demonstrated that IP3R silencing significantly attenuated CIF-induced mitochondrial Ca²⁺ elevation (Figure 11L). Similarly, in the MFN2 overexpression model, IP3R knockdown markedly reduced MFN2-mediated mitochondrial Ca²⁺ increase (Figure 11M,N).

CIF stabilizes MERCs by upregulating MFN2 expression. This stabilization facilitates efficient Ca²⁺ transfer between the ER and mitochondria, preserves the activity of OXPHOS-related enzymes, and enhances mitochondrial oxygen utilization efficiency, thereby promoting ATP production. Consequently, CIF-mediated MFN2 induction improves cardiomyocyte hypoxia tolerance under hypoxic conditions.

3. Discussion

This study provides the first experimental evidence that 16:8 CIF significantly enhances acute hypoxia tolerance in rats. The underlying mechanism involves fasting-induced upregulation of MFN2 expression, which promotes mitochondrial fusion and strengthens the stability of MERCs. This enhanced MERC stability ensures efficient Ca²⁺ transport into mitochondria, thereby maintaining OXPHOS activity, improving mitochondrial oxygen utilization efficiency, and ultimately promoting ATP production to myocardial hypoxia tolerance (Figure 12). Collectively, our findings identify MERCs as a critical structure through which fasting enhances acute hypoxia tolerance, with MFN2 playing a key regulatory role in this process.

Figure 12.

Schematic diagram of CIF stabilizing the structure of MERC through MFN2, ensuring Ca²⁺ transport and ultimately improving hypoxia tolerance under acute hypoxia conditions.

The effects of the 16:8 fasting mode on metabolism, cardiovascular health, and the immune system have been widely studied [12,13,14]. Studies have found that 16-h fasting significantly alters murine hepatic metabolism by triggering rhythmic pathway resonance, activating hepatic proteasomes, and shifting liver metabolism from glucose dependence to fatty acid-derived ketone body utilization. These adaptations also regulate gene expression to facilitate refeeding, with effects being particularly pronounced under alternate-day fasting regimens [15]. Notably, alternate-day time-restricted fasting is well-tolerated in humans, causing minimal discomfort without significant reductions in physical performance. Therefore, this fasting protocol was adopted in this study. Altitudes exceeding 7620 m (25,000 feet) are classified as the ‘death zone’, where alveolar oxygen pressure in humans drops to approximately 30 mmHg, barely sustaining basal metabolic oxygen demands. This places the body at a critical threshold between aerobic oxidation and anaerobic glycolysis, defining it as a severely hypoxic environment [16,17]. Consequently, the time of useful consciousness at 7620 m is commonly used to assess human tolerance to acute high-altitude hypoxia. In this study, we evaluated in vivo hypoxia tolerance using the 24-h survival rate of rats exposed to a simulated altitude of 7620 m. At the cellular level, severe hypoxia was induced in cardiomyocytes by exposure to 1% O₂, with apoptosis rate serving as a cellular hypoxia tolerance indicator.

3.1. CIF Significantly Enhances Hypoxia Tolerance in Rats and Cardiomyocytes

Our findings demonstrate that 16:8 CIF significantly improves the survival rate of rats from 44.4% to 83.3% following acute hypoxia exposure. Additionally, CIF preserves cardiac ejection function and effectively mitigates hypoxia-induced myocardial fiber fragmentation and disarray. At the cellular level, simulated CIF reduces hypoxia-induced apoptosis and mitochondrial ROS production in cardiomyocytes. Fasting also significantly elevates the activity of mitochondrial OXPHOS-related enzymes, which maintains stable oxygen utilization efficiency and ATP production under hypoxic conditions. These observations in rat myocardium suggest that CIF may protect myocardial structure and function from hypoxic damage by preserving mitochondrial OXPHOS in cardiomyocytes and other tissues. This preservation of mitochondrial function likely stabilizes ATP levels, thereby enhancing survival under hypoxia. Thus, the mechanisms by which fasting regulates mitochondrial function deserve further in-depth investigation.

3.2. Fasting Upregulates MFN2 Expression and Enhances the Stability of MERCs

Mitochondrial dynamics play a crucial role in maintaining mitochondrial function and cellular homeostasis. Mitochondria sustain their morphological network and functional integrity through a dynamic process of fission and fusion, enabling adaptation to metabolic fluctuations or cellular stress [18]. For instance, upon exposure to stimuli such as ultraviolet radiation or starvation, mitochondria elongate to enhance the activity of OXPHOS, thereby promoting cell survival and protecting against autophagic degradation [19,20]. Previous studies have shown that starvation promotes mitochondrial fusion, whereas high glucose levels inhibit this process [10,21]. During starvation, elongated mitochondria avoid autophagic degradation and exhibit increased cristae density, enhanced ATP synthase activity, and sustained ATP production [22]. Mitochondrial fusion is primarily mediated by three key proteins: MFN1 and MFN2, which govern outer mitochondrial membrane fusion, and OPA1, which regulates inner mitochondrial membrane fusion. OPA1 exists as two major splice variants, long-form (L-OPA1) and short-form (S-OPA1), and their synergistic interaction is essential for efficient mitochondrial fusion [23]. While MFN1 is mainly localized to the outer mitochondrial membrane, MFN2 is not only present on the outer mitochondrial membrane but also in low abundance on the ER membrane, particularly within MERCs. MFN2 is recognized as a key tethering protein that connects mitochondria and the ER [24]. MERCs are specialized structures formed by tethering mitochondria and the ER via various connector proteins, participating in diverse biological processes including Ca²⁺ and lipid transport, mitochondrial ATP synthesis, and regulation of mitochondrial fission/fusion [8]. The structural characteristics of MERCs are usually described by their number, length, and the intermembrane distance between mitochondria and the ER [25]. In the present study, 16:8 CIF upregulated OPA1 and MFN2 expression in rat myocardium and H9C2 cardiomyocytes, with no significant changes in MFN1 levels. Moreover, CIF induced mitochondrial elongation. The increased MFN2 expression likely contributed to the observed lengthening of MERCs and reduction in distance, thereby enhancing MERC stability; similar results were observed with MFN2 overexpression. It is generally accepted that MFN1, with its higher GTPase activity, plays a central role in driving mitochondrial fusion [18], whereas MFN2, in addition to its role in fusion, is critical for regulating mitochondria-ER contacts, energy metabolism, and cell apoptosis [26]. The differential responsiveness of MFN isoforms to metabolic signals may underlie this expression pattern. Previous investigations have established that mitochondrial adaptation to nutrient stress involves the AMPK-SIRT1 signaling axis [27], with MFN2 being a verified transcriptional target of SIRT1 [28], making it a plausible mechanism for its hypoxia-induced upregulation. In contrast, MFN1 expression demonstrates relative insensitivity to acute metabolic perturbations, as its transcriptional regulation is principally linked to basal mitochondrial homeostasis rather than stress–responsive pathways. Starvation-induced mitochondrial fusion is known to be primarily mediated by OPA1 and MFN1, while MFN2 exhibits broader functionality, including its established role in tethering the endoplasmic reticulum to mitochondria [19]. In contrast, both high-fat diet-fed mice and individuals with type 2 diabetes show reduced expression of MFN2 and OPA1 in skeletal muscle [29,30]. Therefore, we hypothesize that under hypoxic stress, CIF elicits a coordinated adaptive response: it promotes mitochondrial elongation by upregulating OPA1 expression, thereby preventing mitochondrial fragmentation and dysfunction. Concurrently, it enhances MERC coordination and optimizes energy metabolism through increased MFN2 expression, whereas MFN1 levels likely remain relatively stable.

3.3. Fasting Promotes Ca²⁺ Transport Between Mitochondria and the ER via MERCs, Enhances Oxygen Utilization Efficiency, and Facilitates ATP Production in Cardiomyocytes

In aerobic respiration, ATP production primarily occurs through OXPHOS. Multiple enzymes in the mitochondrial electron transport chain (ETC) are regulated by Ca²⁺, including COX IV, the terminal enzyme of the ETC, and CS, a key regulatory enzyme in the tricarboxylic acid (TCA) cycle [31]. In the present study, we found that fasting or MFN2 overexpression significantly increased the enzymatic activity of both COX IV and CS. MERCs serve multiple physiological roles, including the formation of local Ca²⁺ microdomains that facilitate Ca²⁺ transfer. Specifically, Ca²⁺ released from the endoplasmic reticulum (ER) maintains a locally high concentration within a restricted spatial range, enabling efficient uptake by mitochondria [32]. Further examination of mitochondrial Ca²⁺ revealed that fasting or MFN2 overexpression enhanced the transfer of Ca²⁺ from the ER to mitochondria in H9C2 cardiomyocytes, reduced the hypoxia-induced increase in glycolysis level, improved mitochondrial OXPHOS level, and alleviated the hypoxia-induced decrease in COX IV and CS enzyme activities. Mitochondria are the primary source of intracellular ROS. Hypoxia is known to increase mitochondrial ROS production, reduce ATP synthesis, and impair oxygen utilization efficiency [33,34]. Our results confirm these observations, and notably, cells subjected to simulated fasting conditions showed a reduction in mitochondrial ROS production and a significant increase in ATP production under hypoxia. Similarly, MFN2 overexpression decreased ROS levels and improved ATP generation under hypoxia, though the overall ATP levels in the overexpression group still lower than in the fasting group. This discrepancy may result from adenovirus-induced metabolic alterations, since viral infections can modulate host metabolism, diverting ATP towards antiviral responses [35]. The precise mechanism warrants further investigation.

This study, through a gain-of-function approach, demonstrates that MFN2 overexpression mimics the protective effects of CIF (chronic intermittent hypoxia) on cardiomyocytes, suggesting a critical role for MFN2 in this process. However, we observed a concurrent upregulation of the mitochondrial fusion protein OPA1 under both CIF treatment and MFN2 overexpression conditions. This finding indicates that the CIF-induced mitochondrial protection may involve a coordinated network of fusion proteins rather than a singular pathway dependent solely on MFN2. While the phenotypic resemblance between MFN2 overexpression and CIF is striking, it is important to acknowledge that genetic overexpression strategies can indirectly influence other pathways, such as OPA1, through non-specific cellular effects. To more precisely dissect the individual contributions of MFN2 and OPA1 in CIF, future studies could employ temporally specific or conditionally specific genetic manipulation models. In summary, our work establishes MFN2 as a key mediator in CIF-induced cardioprotection, while the integrated regulation of mitochondrial fusion and the cooperative role of OPA1 warrant further investigation.

Building on these mechanistic insights at the molecular level, our study collectively delineates the physiological pathway through which CIF confers hypoxic tolerance. Collectively, our findings indicate that the 16:8 CIF regimen stabilizes MERCs to ensure efficient Ca²⁺ transfer into mitochondria, thereby sustaining the activity of rate-limiting enzymes in OXPHOS, enhancing mitochondrial oxygen utilization efficiency, and ultimately promoting ATP production. This mechanistic insight, which elucidates how CIF improves cellular bioenergetics under hypoxia, extends our previous observation that a 3-day fasting protocol enhances survival in hypoxic rats [7]. Importantly, compared to prolonged fasting, the 16:8 CIF regimen presents a more practical and sustainable intervention, offering a novel and feasible strategy to enhance acute hypoxic tolerance in vivo. While our study demonstrates a protective effect of fasting against acute hypoxia in vivo, the contribution of systemic adaptations (e.g., in stress hormone levels, metabolic state, or organ function) cannot be excluded. To distinguish between systemic and cell-autonomous mechanisms, we employed a controlled in vitro model using H9C2 cells. In this system, which eliminates the influence of circulating factors and neural inputs, fasting mimicry successfully recapitulated key protective phenotypes, including reduced apoptosis, lower ROS levels, and preserved OXPHOS enzyme activity. This provides strong evidence that cardiomyocytes possess an intrinsic, cell-autonomous response to nutrient deprivation, which likely acts in parallel or synergy with systemic effects in the whole animal.

Furthermore, exploring drugs that target the stability of the MERCs structure to rapidly and effectively improve the body’s acute hypoxia tolerance represents a promising direction for translational research. Studies have demonstrated that estrogen can activate the PI3K-AKT axis and preserve mitochondrial integrity to counteract ischemic stress, thereby improving cardiac function [36]. Therefore, only male rats were used in the present study. This choice eliminated the confounding variable of cyclical estrogen levels in female animals but concurrently limits the generalizability of our findings to female subjects. Future work should compare male and female animals to determine whether the enhanced fasting-induced hypoxia tolerance is associated with estrogen.

4. Materials and Methods

4.1. Experimental Animals

Male Sprague-Dowley (SD) rats aged 6–8 weeks were obtained from the Experimental Animal Center of Air Force Medical University. Rats were housed under SPF conditions (temperature: 25 ± 2 °C; humidity: 50–60%; 12 h light/dark cycle) and randomly divided into four groups in a 2 × 2 factorial design, based on oxygen condition (normoxia vs. hypoxia) and feeding regimen (control vs. CIF). Normoxia + Control (N + Con): Rats were housed under normoxic conditions at ground level with free access to food and water. Normoxia + CIF (N + CIF): Rats were housed under normoxic conditions and subjected to the CIF regimen (rats were fasted from 08:00 to 24:00 daily, followed by ad libitum feeding from 24:00 to 08:00 the next day; thereafter, rats were given unrestricted access to food for a consecutive 24 h. This regimen was repeated for two cycles). Hypoxia + Control (H + Con): Following the same ad libitum feeding as the N + Con group, rats were exposed to a hypoxic environment simulating an altitude of 7620 m for 24 h. Hypoxia + CIF (H + CIF): After 48 h of CIF pretreatment as described in the N + CIF group, rats were immediately exposed to a hypoxic environment simulating an altitude of 7620 m for 24 h. All rats had free access to water. The experimental protocol and ethics were approved by the Animal Care Facility of Air Force Medical University (No. 20230017).

4.2. Simulation of Hypobaric Hypoxia Environment

The animal hypoxia chamber developed by our laboratory was used to simulate atmospheric pressure at an altitude of 7620 m. Rats were placed in the chamber, which was depressurized at a rate equivalent to 10 m/s ascent until reaching 7620 m, and maintained for 24 h. The chamber ventilation rate was set to 1 L/min per rat to ensure adequate gas exchange. SiO₂ was placed inside the chamber to adsorb excess water vapor. After hypoxia exposure, the chamber was repressurized to ground level pressure at a rate of 15–20 m/s.

4.3. Cell Culture

The H9C2 cell line (Cellverse, Shanghai, China) was cultured in 1.0 g/L glucose DMEM, supplemented with 10% fetal bovine serum (FBS) at 37 °C under 5% CO₂. The simulated fasting medium contained 0.5 g/L glucose, which was a 1:1 mixture of glucose-free DMEM basal medium and DMEM basal medium containing 1 g/L glucose, supplemented with 0.5 g/L mannitol to adjust the osmotic pressure and 10% FBS.

For the simulated CIF group, cells were subjected to fasting medium for 16 h, then replaced with normal medium for 8 h. On the next day, cells were maintained in normal medium for 24 h. This cycle was repeated on the third day to complete two fasting cycles. For acute hypoxic conditions, cells were exposed to 1% O₂ for 24 h in an anoxic workstation (ELECTROTEK, London, UK).

4.4. Adenovirus Overexpression of MFN2

H9C2 cardiomyocytes were infected with an adenovirus carrying the MFN2 overexpression construct (MFN2-OE) or a control adenovirus (HanBio, Shanghai, China) as previously described [37]. The culture medium was removed and replaced with fresh medium 10–16 h after adenovirus infection. Transfection efficiency was evaluated by Western blotting 48 h post-infection. For acute hypoxia treatment, cells were transferred to 1% O₂ for 24 h after 24 h of adenovirus infection.

4.5. Measurement of Cardiac Function

M-mode echocardiography examination was performed on the rat heart. Rats were anesthetized with 3% isoflurane and maintained at 1–1.5%. After shaving the hair on left thoracic wall, ultrasonic coupling gel was applied, and cardiac function was evaluated using a small animal ultrasound imaging system (Vinno Corp., Suzhou, China). The heart rate (HR), left ventricular diastolic diameter (LVIDd) and left ventricular systolic diameter (LVIDs) were recorded. Cardiac function indexes were calculated using the following formula: LVEDV = 4/3 × π × (LVIDd/2)³, LVESV = 4/3 × π × (LVIDs/2)³, LVEF = (LVEDV − LVESV)/LVEDV × 100%, LVFS = (LVIDd − LVIDs)/LVIDd × 100%.

4.6. Hematoxylin–Eosin Staining

The myocardium of the rats was rapidly collected, rinsed with normal saline to remove blood, and then fixed with 4% paraformaldehyde for 24 h at room temperature. The tissues were subsequently embedded, sectioned, deparaffinized, hydrated, and subjected to hematoxylin and eosin staining. Images were acquired using a light microscope (Olympus, Tokyo, Japan). The degree of myocardial injury was evaluated using an injury grading score (grades 0–4), as previously described [38].

4.7. TUNEL Staining

The myocardium of the rats was rapidly collected, rinsed with normal saline to remove blood, and then fixed with 4% paraformaldehyde at room temperature for 24 h. After paraffin embedding and sectioning, staining procedures were performed according to the instructions of the TUNEL kit (12156792910, Roche, Basel, Switzerland). For co-staining, wheat germ agglutinin (WGA; L4895, Sigma-Aldrich, St. Louis, MO, USA) was used to label cardiomyocytes. Finally, microscopic examination was performed and pictures were collected.

4.8. DHE Staining

The myocardium of rats was collected quickly, and the surface was wiped with filter paper if contaminated. The tissues were frozen in liquid nitrogen for 10–20 s to form blocks, followed by frozen sectioning. Staining procedures were performed according to the instructions of the dihydroethidium (DHE) detection kit (50130ES72, Yeasen Biotechnology Co., Ltd., Shanghai, China). Finally, images were captured using a fluorescence photomicroscope (Olympus, Tokyo, Japan).

4.9. Western Blotting

To obtain protein lysates, cardiomyocytes and rat myocardial tissues were lysed using M-PER^TM Mammalian Protein Extraction Reagent (78051, Thermo Fisher Scientific, Waltham, MA, USA) and T-PER^TM Tissue Protein Extraction Reagent (78510, Thermo Fisher Scientific), respectively. Both reagents were supplemented with a phosphatase and protease inhibitor cocktail (78442, Thermo Fisher Scientific). Protein quantification was performed using the BCA Protein Quantification kit (23225, Thermo Fisher Scientific). Western blotting was subsequently performed as previously described [39]. Membranes were imaged using a chemiluminescence imaging system (ChemiScope 6200, Clinx Science Instruments Co., Ltd., Shanghai, China), and gray values were analyzed using ImageJ software (v1.53t, NIH, Bethesda, MD, USA). Information on antibodies used in this experiment is provided in the Supplementary Tables S1–S4.

4.10. Determination of ATP Level

The rat myocardial tissues were weighed, and cultured cardiomyocytes were collected by digestion and centrifugation. For ATP content determination, samples were processed according to the manufacturer’s instructions of the ATP Assay Kit (E-BC-F002, Elabscience Biotechnology Co., Ltd., Wuhan, China). The fluorescence value of the samples was measured using a multimode microplate reader (SPARK, TECAN, Männedorf, Switzerland), and ATP content in samples was calculated based on the standard curve fitting.

4.11. Flow Cytometry for Cell Apoptosis and Mitochondrial ROS Detection

For apoptosis analysis, cells were stained with the Annexin V-FITC/PI Apoptosis Detection Kit (556547, BD Biosciences, Franklin Lakes, NJ, USA) according to the manufacturer’s protocol. Briefly, cells were incubated with PI- and FITC-conjugated Annexin V for 15 min at 37 °C. Mitochondrial reactive oxygen species (ROSs) were detected using MitoSOX^TM Red (M36007, Thermo Fisher Scientific) by incubating cells at 37 °C for 30 min. After staining, cells were digested, centrifuged, and rinsed with ice-cold PBS. Flow cytometry analysis was performed using a CytoFLEX flow cytometer (Beckman Coulter, Brea, CA, USA), and data were analyzed with FlowJo software (Version 10.8.1, BD Life Sciences, Ashland, OR, USA).

4.12. Transmission Electron Microscope (TEM)

Fresh rat myocardium was fixed in 2.5% glutaraldehyde at 4 °C for 24 h. After hardening, tissues were trimmed into 1 × 1 × 3 mm³ pieces. Then, they were placed in 2.5% glutaraldehyde and continued to be fixed at 4 °C for 24 h. The cultured cardiomyocytes were digested, centrifuged, collected, and fixed with 3% glutaraldehyde at 4 °C. Then subsequent steps were performed as previously described. Images were acquired using TEM (HT7800, Tokyo, Japan). MERCs were identified, and their length and intermembrane distance were measured using ImageJ software.

4.13. Immunofluorescence Staining

Cells were seeded onto confocal culture dishes. After completing the respective treatments for each group, cells were washed twice with PBS and fixed with 4% paraformaldehyde in PBS for 10 min at room temperature. After two washes with PBS, cells were permeabilized with 0.1% Triton X-100 in PBS for 5 min. Subsequently, cells were washed with PBS and blocked with 5% normal goat serum in PBS for 30 min at room temperature. After removal of the blocking solution, cells were incubated with primary antibodies diluted in antibody dilution buffer overnight at 4 °C. The following primary antibodies were used: mouse anti-MFN2 (1:250 dilution; Proteintech, 67487-1-Ig), rabbit anti-PTPIP51 (1:400 dilution; Proteintech, 20641-1-AP), and rabbit anti-VAPB (1:200 dilution; Proteintech, 14477-1-AP). The following day, cells were washed three times with PBS (5 min per wash) and then incubated with species-specific secondary antibodies diluted in antibody dilution buffer for 1 h at room temperature in the dark. The secondary antibodies were donkey anti-mouse IgG conjugated to Alexa Fluor 488 (1:200 dilution; Yeasen, 34106ES60) and donkey anti-rabbit IgG conjugated to Alexa Fluor 594 (1:200 dilution; Yeasen, 34212ES60). Following three final washes with PBS, nuclei were counterstained by applying approximately 100 µL of an antifade mounting medium containing DAPI (Yeasen, 36308ES11). Fluorescence images were acquired using an FV3000 laser scanning confocal microscope (Olympus, Tokyo, Japan).

4.14. MERCs Number Measurement

Cells were co-stained with 250 nM MitoTracker Deep Red FM (M22426, Thermo Fisher Scientific) and 1 μM ER-Tracker^TM Green (E34251, Thermo Fisher Scientific) at 37 °C for 30 min in the dark. Nuclei were labeled with Hoechst 33342. Then the staining solution was removed and cells were washed twice with preheated fresh medium. Images were acquired using a high-content imaging system (Operetta CLS^TM, PerkinElmer, Waltham, MA, USA). The Pearson correlation coefficient was calculated using Fiji software (v2.3.0, based on ImageJ; NIH, USA) to quantify mitochondria-ER colocalization.

4.15. Ca²⁺ Detection

Rhod-2 (R1245MP, Thermo Fisher Scientific) was used to label mitochondrial Ca²⁺, and mitochondria were co-stained with MitoTracker Green^TM (M7514, Thermo Fisher Scientific). Both probes were dissolved in anhydrous DMSO. After treatment, cells were incubated with Rhod-2 at a final concentration of 5 μM and MitoTracker Green^TM at 100 nM in the dark at 37 °C under 5% CO₂ for 30 min, followed by two washes with preheated PBS to remove excess probes. Images were acquired using a high-content imaging system (Operetta CLS^TM, PerkinElmer, Waltham, MA, USA), and the Pearson correlation coefficient was calculated to assess the colocalization between Ca²⁺ signals and mitochondria, which indirectly reflects the extent of Ca²⁺ influx. For inositol 1,4,5-trisphosphate receptor (IP3R) inhibition, 50 µM 2-Aminoethoxydiphenyl borate (2-APB) was added to the culture medium throughout the experiment.

4.16. Enzyme Activity Measurement

Myocardium was homogenized in lysis buffer, centrifuged, and the supernatant was collected. For cardiomyocytes, cells were trypsinized, harvested, and lysed by sonication in lysis buffer, followed by centrifugation to collect the supernatant. Subsequent steps were performed according to the manufacturer’s instructions for the rat mitochondrial respiratory chain complex IV (COX IV) ELISA kit (F0311-RA, Fankew, Shanghai, China) and the rat citrate synthase (CS) ELISA kit (F40335-A, Fankew, Shanghai, China). Briefly, the microplate was set up with designated wells for samples and standards. After adding the samples and a serial dilution of standards to their respective wells, horseradish peroxidase (HRP)-conjugate reagent was added to all wells. The plate was sealed and incubated at 37 °C for 60 min. Following incubation, the wells were aspirated and washed five times with wash buffer. Then, the substrate solution was added, and the plate was incubated at 37 °C in the dark for 15 min. The enzymatic reaction was stopped by adding the stop solution. The optical density (OD) at 450 nm was immediately measured using a Multiskan MS microplate reader (Thermo Fisher Scientific, Vantaa, Finland), and the enzyme activity in the samples was calculated based on the standard curve fitting.

4.17. Oxygen Consumption Rate (OCR) and Extracellular Acidification Rate (ECAR) Analysis

Mitochondrial respiration was analyzed using a Seahorse XFe96 Extracellular Flux Analyzer (Agilent Technologies, Santa Clara, CA, USA) as previously described [40]. Briefly, cells were seeded at a density of 10⁴ cells per well in Seahorse XF96 microplates and incubated overnight at 37 °C with 5% CO₂. After the determination of basal respiration, oligomycin at 1.0 μM and FCCP at 1.5 μM were added to determine proton leakage and maximum respiratory capacity, respectively. Finally, antimycin A and cyanide were added to measure spare respiratory capacity. ECAR was detected by adding glucose, hypomycin, and 2-deoxyglucose in a glucose-free medium. All OCR and ECAR values were normalized to total cellular protein content.

4.18. siRNA Transfection

H9C2 cardiomyocytes were seeded in 6-well plates at a density of 2 × 10⁵ cells per well 24 h prior to transfection. The siRNA (General Biol, Anhui, China) was diluted in Opti-MEM^TM medium (31985062, Thermo Fisher Scientific) to a final concentration of 50 nM. Lipofectamine^TM 2000 (11668019, Thermo Fisher Scientific) was also diluted in Opti-MEM^TM medium and incubated at room temperature for 5 min. The diluted siRNA was then mixed with the diluted Lipofectamine^TM 2000 and incubated at room temperature for 20 min to allow complex formation. Then the transfection mixture was added to the cell culture. After 6 h of transfection, the medium was replaced with fresh culture medium. Protein expression levels were analyzed by Western blot 56 h post-transfection. The siRNA sequences targeting the mRNAs corresponding to different isoforms of IP3R are as follows. Itpr1-siRNA sense strand: 5′-GGGACAAGUUUGACAAUAATT-3′; Itpr1-siRNA antisense strand: 5′-UUAUUGUCAAACUUGUCCCTT-3′; Itpr2-siRNA sense strand: 5′-UGAAAGAAGUUGUGAAUCATT-3′; Itpr2-siRNA antisense strand: 5′-UGAUUCACAACUUCUUUCATT-3′; Itpr3-siRNA sense strand: 5′-ACACGGAGGUGGAGAUGAATT-3′; Itpr3-siRNA antisense strand: 5′-UUCAUCUCCACCUCCGUGUTT-3′; NC-siRNA (negative control) sense strand: 5′-UUCUCCGAACGUGUCACGUTT-3′; NC-siRNA (negative control) antisense strand: 5′-ACGUGACACGUUCGGAGAATT-3′.

4.19. Statistical Analysis

Statistical analyses were performed using GraphPad Prism 10.6.0. Two-way ANOVA with multiple comparisons was used for multiple group comparisons in most experiments, and an unpaired t-test was used for two group comparisons. Survival rate differences were analyzed by a chi-square test. Pearson correlation coefficient analysis was performed to assess the colocalization of mitochondria with the endoplasmic reticulum and with Ca²⁺. Values are presented as mean ± SEM with * p < 0.05, ** p < 0.01 or ^# p < 0.05, ^## p < 0.01 considered to be significant and highly significant. All replicates in this study were biological replicates from different samples.

5. Conclusions

In summary, our study reveals that under hypoxic conditions, 16:8 CIF enhances cardiomyocyte hypoxia tolerance by stabilizing MERCs through upregulation of MFN2. This stabilization ensures efficient Ca²⁺ transfer from the ER to mitochondria, maintains the activity of OXPHOS enzymes, and improves mitochondrial oxygen utilization, ultimately boosting ATP production. These findings demonstrate the therapeutic potential of the 16:8 CIF regimen for mitigating hypoxic cardiomyocyte injury and reveal MERCs modulation as a novel approach for cardiovascular interventions.

Abbreviations

The following abbreviations are used in this manuscript:

2-APB	2-Aminoethoxydiphenyl borate
ATP	Adenosine triphosphate
COX IV	Cytochrome c oxidase subunit IV
CS	Citrate synthase
DHE	Dihydroethidium
DMEM	Dulbecco’s modified eagle medium
DMSO	Dimethyl sulfoxide
DRP1	Dynamin-related protein 1
ECAR	Extracellular acidification rate
ER	Endoplasmic reticulum
FCCP	Carbonyl cyanide-4- (trifluoromethoxy)phenylhydrazone
HACE	High-Altitude Cerebral Edema
HAPE	High-Altitude Pulmonary Edema
HBSS	Hanks’ Balanced Salt Solution
IP3R	Inositol 1,4,5-trisphosphate receptor
LVEDV	Left ventricular end-diastolic volume
LVEF	Left ventricular ejection fraction
LVESV	Left ventricular end-systolic volume
LVIDd	Left ventricular diastolic diameter
LVIDs	Left ventricular systolic diameter
LVSF	Left ventricular shortening fraction
MERC	Mitochondria–endoplasmic reticulum contact
MFN1	Mitofusin1
MFN2	Mitofusin2
MOI	Multiplicity of infection
OCR	Oxygen consumption rate
OPA1	Optic atrophy 1
OXPHOS	Oxidative phosphorylation
p-DRP1	Phospho-mitochondrial dynamics-related protein 1
PTPIP51	Protein tyrosine phosphatase interacting protein 51
ROS	Reactive oxygen species
S-OPA1	Short form of optic atrophy 1
TUNEL	Terminal deoxyribonucleotidyl transferase dUTP nick-end labeling

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/ijms27052117/s1.

Author Contributions

Conceptualization, X.C. and Z.Y.; cellular experiments and formal analysis, X.C. and B.J.; animal experiments, T.X. and X.C.; writing—original draft preparation, X.C. and T.X.; writing—review and editing, M.X. and Z.Y.; funding acquisition, Z.Y. All authors have read and agreed to the published version of the manuscript.

Institutional Review Board Statement

The animal study protocol was approved by the Laboratory Animal Welfare and Ethics Committee of Air Force Medical University (protocol code 20230017, approval date: 3 March 2022).

Informed Consent Statement

Not applicable.

Data Availability Statement

The original contributions presented in this study are included in the article/Supplementary Materials. Further inquiries can be directed to the corresponding author.

Conflicts of Interest

The authors declare no conflicts of interest.

Funding Statement

This research was funded by the National Natural Science Foundation of China (NSFC), grant number 32371237.