Spermidine Alleviates Intrauterine Hypoxia-Induced Offspring Newborn Myocardial Mitochondrial Damage in Rats By Inhibiting Oxidative Stress And Regulating Mitochondrial Quality Control Part 1

Abstract

Background: Intrauterine hypoxia (IUH) increases the risk of cardiovascular diseases in offspring. As a reactive oxygen species (ROS) scavenger, polyamine spermidine (SPD) is essential for embryonic and fetal survival and growth. However, further studies on SPD protection and mechanisms for IUH-induced heart damage in offspring are required. 

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Objectives: This study aimed to investigate the preventive effects of prenatal SPD treatment on IUH-induced heart damage in newborn offspring rats and its underlying mitochondrial-related mechanism. 

Methods: The rat model of IUH was established by exposure to 10% O2 seven days before the term. Meanwhile, for seven days, the pregnant rats were given SPD (5 mg.kg-1.d-1; ip). The one-day offspring rats were sacrificed to assess several parameters, including growth development, heart damage, cardiomyocytes proliferation, myocardial oxidative stress, cell apoptosis, and mitochondrial function, and have mitochondrial quality control (MQC), including mitophagy, mitochondrial biogenesis, and mitochondrial fusion/fission. In in vitro experiments, primary cardiomyocytes were subjected to hypoxia with or without SPD for 24 hours. 

Results: IUH decreased body weight, heart weight, cardiac Ki67 expression, the activity of SOD, and the CAT and adenosine 5’-  triphosphate (ATP) levels and increased the BAX/BCL2 expression and TUNEL-positive nuclei numbers. Furthermore, IUH also caused mitochondrial structure abnormality, dysfunction, and decreased mitophagy (decreased number of mitophagosomes), declined mitochondrial biogenesis (decreased expression of SIRT-1, PGC-1α, NRF-2, and TFAM), and led to fission/fusion imbalance (increased percentage of mitochondrial fragments, increased DRP1 expression, and decreased MFN2 expression) in the myocardium. Surprisingly, SPD treatment normalized the variations in the IUH-induced parameters. Furthermore, SPD also prevented hypoxia-induced ROS accumulation, mitochondrial membrane potential decay, and the mitophagy decrease in cardiomyocytes. 

Conclusion: Maternal SPD treatment caused IUH-induced heart damage in newborn offspring rats by improving the myocardial mitochondrial function via anti-oxidation and anti-apoptosis, and regulating MQC.

1. Background

Epidemiological and animal studies indicate that an adv se intrauterine environment is associated with an increased risk of cardiovascular diseases in adulthood (1). Prenatal hypoxia, the most common adverse intrauterine environment, can make the fetus unable to realize its genetically-determined growth potential, manifested as growth retardation and organ function injury in ooff-spring(2). Chronic fetal hypoxia often results from pregnancy with increased placental vascular resistance such as pre-clampsia, inter-pregnancy at high altitude, or maternal respiratory diseases. More significantly, one-third of them are affected by obstructive sleep apnea hypoventilation syndrome (OSAHS) in late pregnancy, and it is also associated with the development of gestational hypertension syndrome. The most critical pathophysiological changchangeSAHS is chronic intermittent hypoxia (3, 4). In several animal models, intrauterine hypoxic (IUH)  lead to cardiac efficiency reduction, diastolic and systolic dysfunction, hypertrophic growth, decreased cardiomyocyte proliferation, and delayed cardiomyocyte maturation in the fetal and neonatal heart, thereby increasing the risk of heart disorders in adult offspring (5). The programming effect of chronic hypoxia can mainly explain the mechanism at critical stages of the development of the heart, which leads to cardiac oxidative stress enhancement and cell apoptosis increase in offspring (6-9). The con pt of developmental programming makes sense because our physiology is much more malleable and plastic during early life. The intrauterine condition determines and programs the concepts of physiology and metabolism that our life. Accordingly, the fetal adaptive response to IUH leads to the development of adult cardiovascular diseases (10). Mitochondria are complex organelles playing crucial roles in cellular energy-generating and a myriad of cellar signaling events. The structural and functional integrity of mitochondria is critical for the survival of cardiomyocytes, and when compromised, the disruption of mit-hondrial homeostasis results in the development of carac diseases. Recently, several studies have focused on the role of chronic prenatal hypoxia-inducing mitochondrial dysfunction in mediating offspring heart damage. It has been reported that that mitochondrialiratory function was impaired, and the expression of several mitochondrial mol rules altered the perinatal heart exposed to IUH (11). Moreover, IUH leads to decreased mitochondrial complex enz e activity and slice dysfunction of the heart in ult offspring following myocardial ischemia (12). Accordingly, this study aimed to discover the molecular link amo prenatal hypoxia, oxidative stress, mitochondrial dys action, and heart injury in new offspring (13).

The development and maturation of this high-volume mitochondrial system in the myocardium mainly occur in the perinatal and postnatal development stages, primarily due to the cardiac metabolic shift from using glucose o fatty acids for the adenosine 5’-triphosphate (ATP) generation after birth (14). Emerging evidence suggests mitochondrial quality control (MQC) mechanisms are critical determinants for the maturation of cardiomyocytes (15). Mitochondrial quality control, mainly including mitochondrial biogenesis, mitophagy together with dynamics, a finely tuned regulatory network, orchestrates the quantity and quality of mitochondria and improves mitochondrial function and cardiomyocyte survival under stress conditions (16). Peroxisome proliferators-activate receptors γ (PPARγ) coactivator 1 (PGC-1) play a critical role n driving mitochondrial biogenesis and function in the heart (17). Peroxisome proliferators-activate receptors γ  coactivator 1 α/β (-/-) mice hearts exhibit the signatures f the maturational defect and severe abnormality in mitochondrial function and density (18). Furthermore, deleting the mitophagy-mediator Parkin prevents morphological and functional mitochondria maturation in the neonatal stage (19). Mitochondria are highly dynamic organelles,  undergoing constant fission and fusion events associated with their function. At the late embryonic period, the car ac-specific genetic ablation of mitochondrial fusion pro in MFN1 and MFN2 in mice display severe mitochondrial dysfunction after birth and develop cardiomyopathy (20). Strikingly, a recent study revealed that chronic hyp ia disrupted mitochondrial dynamics in the fetal gui a pig forebrain (21). Accordingly, MQC may act as a focal point in myocardium injury development in the IUH neonatal offspring. However, little is known about the effects of prenatal hypoxia on the neonatal cardiac MQC system and the effect of MQC dysfunction on neonatal heart health. This study took over this mission to explore the mitochondrial-related mechanism of IUH-induced myocardial damage in new offspring and further explore preventative strategies to reduce the cardiac injury of IUH.

Polyamines (PAs), including putrescine (PU), spermidine (SPD), and spermine (SP), are present in nearly all living organisms and are essential for embryonic and fetal sur val, growth, and development (22-24). Studies found that sheep exposed to IUH can improve embryonic dysplasia by supplementing exogenous polyamines (25, 26). Furthermore, the growing body of literature indicates the role of polyamines in scavenging free radicals and protecting DNA, proteins, and lipids from the detrimental damage of oxidative stress. It is also revealed that polyamine supplementation increases the life span in model organisms via antioxidant, anti-inflammatory, and mitophagy-inducing pro-tries (27-29). Recent studies have documented that sup mentation with SPD in dietary is cardioprotective and prolongs lifespan in both mice and humans by stimulating mitophagy and mitochondrial respiration and improving heart function (30, 31). More importantly, we previously reported that maternal hypoxic exposure during the late stages of fetal development resulted in the dased ana list and increased catabolism of polyamines in the carac tissue of newborn rats. SPD prevented heart injury in ven-day offspring rats exposed to IUH by inhibinhibiting mitochondrial mentation (32).

2. Objectives 

In this study, IUH is hypothesized to induce mitochondrial structure and function deficits by increasing oxidative stress and destroying the MQC mechanism in the newborn offspring’s heart, and maternal SPD treatment in utero is assumed to reduce myocardial injury by decreasing the development program of mitochondria. This study may contribute to the development of preventative or therapeutic strategies for IUH offspring to prevent adult cardiovascular diseases.

3. Methods

3.1. Animals 

The male and female Wistar rats (3 months old) were purchased from the Department of Laboratory Animals at Harbin Medical University. All procedures were approved by the Ethics Review Committee of Harbin Medical University (China), and all experiments were conducted by the National Institutes of Health guidelines. The rats with a male-to-female ratio of 2: 1 were randomly placed in a cage for mating. Vaginal smears were performed the following day to detect the presence of sperm in vaginal plugs or vaginal smears,  which was confirmed as the zero-day of pregnancy. The pregnant rats were kept in a room with controlled humidity (60%) and controlled temperature (21°C), and the light-dark cycle was 12: 12 hours.

3.2. Intrauterine Hypoxia Model 

From the 15th to the 21st day of pregnancy, the rats in the hypoxia group (n = 10) were put into a closed plexiglass chamber, injected with air and nitrogen, and monitored by an oxygen analyzer (Pro OX120; BioSpherix, New York, USA),  and inhaled with an oxygen content of 10% for four hours per day. The arterial blood samples were taken from the right femoral artery, the blood gas and pH values were measured to maintain the arterial oxygen partial pressure of 50 - 55 mmHg, the blood oxygen saturation was maintained at 80 - 85%, and specific procedures were performed as previously described (32). The experimental female rats were randomly divided into four groups: control group (control), intrauterine hypoxia group (Hpx), intrauterine hypoxia + spermidine group (Hpx-Spd), and the intrauterine hypoxia + spermidine + inhibitor group (Hpx-Spd-DFMO). Six rats per group were intraperitoneally injected on the 15th - 21st day of pregnancy. The rats were given 0.9%  saline (1 mL/kg/d) in the control and Hpx groups, SPD (5  mg/kg/d) in the Hpx-Spd group, and SPD (5 mg/kg/d) and difuoromethy-L-ornithine (DFMO, an inhibitor of the key enzyme of polyamine synthesis ODC) (5 mg/kg/d) in the Hpx-Spd-DFMO group, respectively. After delivery, 1-day-old  newborns were sacrificed, and their hearts were extracted for follow-up experimental studies.

3.3. Histological Analysis 

The left ventricular tissue of the rats was cut into a  thickness of < 1 cm, fixed with 4% paraformaldehyde, dehydrated with alcohol, and embedded in paraffin. The embedded paraffin was cut into 5 mm thick slices, then dried at a constant oven temperature of 60°C, dewaxed with xylene, and followed by staining hematoxylin-eosin (HE). The tissue slices were observed to assess the changes in cardiac morphology and structures with an optical microscope (Eclipse E200; Nikon, Tokyo, Japan).

3.4. Immunofluorescence Analysis 

Ki67 immunofluorescence staining was performed as previously described (33). Briefly, the left ventricular tissue of the rats was fixed in 4% formalin, embedded in paraffin;  dewaxing, hydration, and antigen repair were first completed. Those tissues were blocked with 0.5% bovine serum albumin for 2 hours and then incubated with Ki67 Rabbit Monoclonal Antibody (1: 100, AF1738, Beyotime, China) at 4°C overnight. After washing with PBS, the tissue was incubated with Alexa fluor labeled Goat anti-rabbit IgG (1: 500, A 0468, Beyotime, China) and counterstained with DAPI for nuclei. The images were viewed and scanned under laser confocal microscopy (OLYMPUS, FV1000, Japan). The software was used to analyze the colocalization of the merged images.

3.5. Quantification of Fibrosis 

Cardiac fibrosis was assessed by Masson’s trichrome staining. As described above, the cardiac tissue sections from the neonatal rats were dewaxed and hydrated by standard methods and then stained with Masson’s trichrome according to the protocols. Two nonadjacent cross-sections were used for each heart. The percentage of fibrotic area in the total left ventricular myocardial area was analyzed using ImageJ software, vl.52 (NIH, Bethesda, MD).

3.6. TdT-Mediated dUTP Nick End Labeling and Apoptotic Cell Measurements

A TdT-mediated dUTP nick end labeling (TUNEL) assay was used to determine the number of apoptotic cells in the one-day-old neonatal rat hearts. The heart tissues were treated following our previously described procedure using a Cell Death Detection Kit (Roche, Germany) according to the manufacturer’s instructions. Three slides from each block were evaluated for the percentage of apoptotic cells. Four slide fields were randomly examined with 200×magnification. In total, 100 cells were counted in each field.

3.7. Measurement of Antioxidant Enzyme Activity

Superoxide dismutase (SOD) and catalase (CAT) activity were measured using commercial kits (SOD: A001-3- 1 and CAT: A007-1-1; Jiancheng Bio. Institute, Nanjing, China) with a spectrophotometer (Perkin- Elmer, Norwalk, CT, United States). According to the manufacturer’s instructions, the operation was completed, and the protein concentration was measured using the bicinchoninic acid method (Pierce, Rockford, United States) with bovine serum albumin (BSA) as a standard (34).

3.8. Measurement of Adenosine 5’-Triphosphate Content

The ATP content in cardiac tissue was measured using an ATP assay kit (S0026B, Beyotime, Bio. Institute, China). According to the manufacturer’s instructions, the lysate was added in proportion according to the tissue weight,  homogenized with a glass homogenizer, and then centrifuged at 4°C 12000 g. The supernatant was taken, and the content of ATP in each sample was detected with a luminometer (NanoDrop, Nanodrop2000, Thermo, USA) BCA  kit (P0012s, Beyotime, Bio. Institute, China). The bicinchoninic acid method was used to determine the protein concentration and then convert the concentration of ATP  into nmol/g protein.

3.9. Transmission Electron Microscopy

The cardiac apical tissue was dissected into approximately 1 mm × 1 mm × 1 mm small pieces and then fixed in glutaraldehyde phosphate buffer at 4°C. After routine dehydration, soaking, embedding, and staining, the ultrathin sections of 50 - 70 nm were made. The ultrastructure of the cardiac tissue was observed under a transmission electron microscope (TEM) and photographed (H600 Hitachi, Tokyo, Japan). Single mitochondria and myofilaments were mapped under the condition of 10000 times magnification with Image J software version 1.80 (National Institutes of Health), and their areas were measured from each heart (35). Meantime, mitochondrial fragments < 1 µm3, which were not divided (usually round), were identified, and the average percentage of mitochondrial fragments in the view field was counted using the Mitochondrial Fragmentation Index (MFI).

3.10. Mitochondrial Isolation 

Mitochondria were isolated at 4°C using differential centrifugation with a Mitochondria Isolation Kit (Beyotime Biotechnology, Shanghai, China). Briefly, fresh heart tissue was cut into tissue pieces and centrifuged with 10  volumes of pre-chilled PBS, the supernatant was discarded,  the precipitate was digested with trypsin for 20 min, the added isolation buffer A was centrifuged, the supernatant was transferred to another tube, centrifuged again, and the precipitated fraction was the isolated mitochondria. The final cardiac mitochondrial pellet was resuspended in homogenizing buffer, stored on ice, and used for experiments on mitochondrial respiration function within 4h.

3.11. Measurement of Mitochondrial Oxygen Consumption

Mitochondrial oxygen consumption was measured by a Clark-type oxygen electrode (Hansatech Instruments, Norfolk, UK) in a mitochondrial respiration buffer. Pyruvate (5 mM) and malate (5 mM) were used as a substrate for complex I-containing mitochondria at the final concentration of 500 µg protein/mL. ADP-stimulated oxygen consumption (state 3 respiration) was measured in the presence of 200 µM ADP, and ADP-independent oxygen consumption (state 4 respiration) was monitored. The respiratory control ratio (RCR, state 3 divided by state 4) reflects oxygen consumption by phosphorylation (coupling). Procedures continued, as previously described (36).

3.12. Western Blot Analysis

The sample of cardiac tissues was harvested and stored at -80°C. Frozen left ventricular cardiac tissues were homogenized in ice-cold RIPA lysis buffer (Beyotime Inc., Shanghai, China P0013B). The protein concentrations were quantified using a BCA protein assay kit (Beyotime Inc., Shanghai, China P0006C). The samples containing total protein were separated by 10% (w/v) SDS-PAGE and transferred onto a PVDF membrane (Millipore, Bedford, MA, United States). The following antibodies were used: Antibodies for GAPDH (1:2000,10494-1-AP), MFN2 (1:1000,12186-1- AP), SIRT-1 (1:1000,13161-1-AP), NRF-2 (1:600,16396-1-AP), TFAM (1:1000,19998-1-AP), and BAX (1.1000,509599-2-Ig) were purchased from Proteintech (WuHan, China), BCL2 (1: 2000,  sc-7382) and DRP1 (1:1000, sc-271583) were purchased from Santa Cruz Biotechnology (Dallas, TX) and PGC-1α (1:1000,  ab106814, Abcam, Cambridge, MA, UK). The secondary antibody (horseradish peroxidase-labeled goat anti-rabbit IgG)  was from Beyotime Corporation (Shanghai, China). The intensities of the protein bands were quantified using a Fluor Chem Chemiluminescence gel imaging system (Protein Simple, USA). The optical density of the protein bands was analyzed with Image J software version l.52 (NIH, Bethesda, MD).

3.13. Hypoxic Cardiomyocyte Model

Neonatal rat cardiomyocytes (NRMCs) were isolated and cultured by standard methods, as previously described (32). Briefly, the hearts of the three-day neonatal rats were extracted, minced, cultured, and then digested in 0.25% trypsin and 0.02% EDTA (Beyotime Biotechnology, Shanghai, China). After centrifugation, the precipitates were transferred to DMEM supplemented with 10% fetal calf serum (Biolot, Russia) and incubated in humid air containing 5% 2. Three days after being seeded, the cells were placed in a glass hypoxic chamber (Biospherix OxyCycler C42, Redfield, NY), and filled with nitrogen for 8 min to discharge the residual oxygen. These cardiomyocytes were randomly divided into the following groups: (1) Control group (control), cells cultured under normal incubation conditions; (2) hypoxia (Hpx) group, cells placed in the hypoxic chamber for 24 h and then cultured normally for 24  h; (3) Hpx-Spd group, cells placed in the hypoxic chamber and incubated with 10 µmol/L SPD for 24 h; (4) Hpx-SpdDFMO group, cells placed in a hypoxic chamber and incubated with 10 µmol/L SPD + 2 mmol/L DFMO for 24h.

3.14. Measurement of Reactive Oxygen Species

The ROS generation was measured by the dihydroethidium (DHE) staining assay (Cat No. S0063, Beyotime, China). Briefly, primary cardiomyocytes were incubated with 5 µmol/L DHE at 37°C for 30 min, washed with PBS, and then moved under the microscope to observe the changes in fluorescence intensity. Images were taken with an Olympus FluoView FV1000 fluorescence microscope (Olympus Optical Co., Ltd., Takachiho, Japan) at an excitation wavelength of 535 nm, and the maximum emission wavelength was 610 nm, n > 20 cells per group.

3.15. Determination of Mitochondrial Membrane Potential

The dye tetramethylrhodamine ethyl ester (TMRE) is positively charged and can be selectively located in mitochondria. It is widely used to determine mitochondrial membrane potential (∆Ψm). In short, TMRE staining working solution at a concentration of 200 µmol/L was added to the grouped treated cardiomyocytes, mixed thoroughly, and placed in a cell incubator at 37°C for 20 min. The supernatant was aspirated, and the cells were washed with PBS and moved to an inverted microscope to observe the change in fluorescence intensity. The excitation wavelength was 549 nm, and the emission light was 579 nm. The red fluorescence intensity indicated the change of ∆Ψm,  n > 20 cells per group.

3.16. Mitochondrial and Lysosomal Localization Experiment

According to the instruction, Mito-Tracker Green (NO.C1048, Beyotime, China) and Lyso-Tracker Red (NO.C1046, Beyotime, China) used the mitochondrial and lysosomal colocalization analysis. NRCMs were loaded with 200 nM MitoTracker Green FM and 50 nM LysoTracker Red in HBSS for 30 min before experiments,  cells were washed with PBS, and then cell images were obtained using an Olympus FluoView FV1000 fluorescence microscope. The 488 nm laser line was used to excite MitoTracker Green fluorescence, measured between 505  and 515 nm. For LysoTracker Red, the 577 nm laser line was used with a measurement of 590 nm. Red and green pixel intensity overlay was determined using quantification software on the Nikon Eclipse Microscope, n > 20 cells per group.

3.17. Statistical Analysis

All data from the experimental groups were compared using one-way ANOVA followed by Bonferroni post hoc test with GraphPad Prism software version 8 (GraphPad Software Inc., La, Jolla.CA) and SPSS software version 17.1 (SPSS, Chicago, IL, United States). Data were expressed as mean ± SEM, and the significance level was set to be P < 0.05.

4. Results

4.1. Offspring and Heart Characteristics

Body weight (BW) and heart weight (HW) were measured, and the HW to BW ratio (HW/BW) was calculated (Figure 1). The results showed that the newborn rats’ BW  and HW decreased, and their HW/BW increased due to intrauterine hypoxia. Compared to the IUH group, the BW  and HW of neonatal rats in the SPD group increased (P < 0.05), and the HW/BW decreased (P < 0.05); Compared to the SPD group, both BW and HW of the DFMO treatment group decreased (P < 0.05), and HW/BW increased significantly (P < 0.05).

4.2. Effects of SPD on Myocardial Morphological Structure, Cell Proliferation, and Fibrosisin Newborn Offspring Exposed to

The cardiac HE staining results showed that the hearts of one-day offspring exposed to IUH displayed swelling and loosely arranged myocardial fibers. However, the IUH  hearts treated with SPD maintained an acceptable myocardial tissue structure (Figure 2A). Next, we evaluated the number of binuclear cardiomyocytes with the HE-stained tissue slice; the number of binuclear cardiomyocytes was larger in the IUH group than in the control group (P < 0.05). Compared to the IUH group, the proportion of binuclear cardiomyocytes in the SPD treatment group decreased significantly (P < 0.05); DFMO attenuated the effect of SPD (P < 0.05) (Figure 2C). We further detected the expression of Ki67 (a marker of cell proliferating) in rat myocardium using the immunofluorescence method. The higher the expression of Ki67, the stronger the pink fluorescence by merging red and blue (Figure 2E). The results showed that compared to the control group, the expression of Ki67 in the IUH group decreased significantly (P < 0.05), the Ki67 expression significantly increased following the SPD administration (P < 0.05), and the effect of SPD was abolished by DFMO (P < 0.05) (Figure 2F). These results indicate that SPD can inhibit the premature withdrawal of cardiomyocytes from the cell cycle induced by IUH and promote the proliferation of cardiomyocytes in the new offspring exposed to IUH. Then we used Masson staining to detect the changes in myocardial collagen content and assessed myocardial fibrosis by collagen area measurement (Figure 2BandD). We found that myocardial collagen deposition in the hearts of the newborn rats exposed to IUH increased (P < 0.05), the level of which was higher compared to the control group. On the contrary, the area of myocardial fibrosis following the SPD treatment significantly reduced (P < 0.05). However, compared to the SPD treatment group, the fibrosis area significantly increased in the HpxSpd-DFMO group (P < 0.05).

4.3. Effects of Spermidine on Myocardial Mitochondrial Structure, Respiratory Function, and Adenosine 5’-Triphosphate Content in Newborn Offspring Exposed to Intrauterine Hypoxia

The ultrastructural structure changes of cardiac tissue and mitochondrial characteristic were analyzed by TEM (Figure 3A). Image J software was used to quantify the mitochondrial content percentage (mitochondrial area in the whole cell area) and mitochondrial area (Figures 3B and C). The results showed that in the control group, the myocardial myofilaments were orderly arranged, the sarcomere structure was clear, the mitochondria were compact, the matrix was denser, and the mitochondrial cristae were orderly arranged. However, mitochondria swelled, and the loosening matrix and decreased density were observed in some cardiomyocytes of the IUH group. Compared to the control group, the proportion of mitochondria in cardiomyocytes and the area of mitochondria were both reduced (P < 0.05). However, in the rat hearts of the SPD treatment, the myocardial myofilaments were neat, the sarcomere structure was clear, the mitochondrial matrix was compact, and the mitochondrial swelling decreased. Compared to the IUH group, the proportion of mitochondria in cardiomyocytes increased (P < 0.05), and the area of mitochondria increased (P < 0.05). DFMO inhibited these SPD-induced effects (P < 0.05).

We used pyruvate/malate as the substrate to evaluate the mitochondrial respiratory function, including states 3  and 4 respiratory rates and RCR (Figure 3D - F). We noticed that compared to the control group, the respiratory rate in states 3 and 4 and the RCR of the IUH group were all significantly lower (P < 0.05). Interestingly, the RCR of states 3 and 4 recovered following the SPD treatment (P < 0.05). In contrast, these SPD effects were significantly inhibited in the DFMO treatment group (P < 0.05). Similarly, compared to the control group, the myocardial ATP content in the IUH group significantly reduced (P < 0.05). Compared to the IUH group, the ATP content remarkably increased in the SPD-treated group (P < 0.05), and DFMO attenuated the effects of SPD (P < 0.05) (Figure 3G). These findings suggest that SPD can protect the myocardial mitochondrial structure and function damage and prevent the decline of the ATP  levels in neonatal offspring rats exposed to IUH.

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