Spermidine Alleviates Intrauterine Hypoxia-Induced Offspring Newborn Myocardial Mitochondrial Damage in Rats By Inhibiting Oxidative Stress And Regulating Mitochondrial Quality Control Part 2

4.4. Effects of Spermidine on Myocardial Antioxidative Activity  and Apoptosis in Newborn Offspring Exposed to Intrauterine Hypoxia

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We measured the activities of antioxidant enzymes SOD and CAT in the myocardium by colorimetry (Figures 4A  and B). It was found that the activities of SOD and CAT in the IUH group were significantly lower than those in the control group (P < 0.05); Compared to the IUH group, the activities of SOD and CAT in the SPD group increased significantly (P < 0.05), andDFMO abolished the effect of SPD (P < 0.05). Subsequently, we evaluated cell apoptosis by detecting TUNEL-positive nuclei (Figure 4CandD) and the expression of pro-apoptosis protein BAX and anti-apoptosis BCl-2 (Figures 4E and F). We noticed that the number of TUNELpositive cardiomyocytes and the BAX/BCL2 protein expression ratio were substantially higher in the IUH group than those in the control group (P < 0.05). In contrast, the ratio of TUNEL-positive cells and BAX/BCL2 protein expression ratio were lower in the myocardium of SPD-treated rats than in the IUH group (P < 0.05). DFMO eliminated the effect of SPD (P < 0.05). To sum up, SPD given in utero can reverse the decrease of antioxidant enzyme activities and the increase of cell apoptosis in offspring hearts induced by IUH.

4.5. Effects of Spermidine on Production of Reactive Oxygen Species, Mitochondrial Membrane Potential, and Mitophagy in Cardiomyocytes Exposed to Hypoxia

Mitochondrial dysfunction is mainly manifested as an increase in ROS production, a decrease in ATP synthesis, and the accumulation of dysfunctional mitochondria. Mitochondrial membrane potential (∆Ψm) is the proton electrochemical gradient (H+ concentration gradient and transmembrane potential difference) across the mitochondrial inner membrane, which is formed during the electron transfer of the mitochondrial oxidative respiratory chain, in which a small number of ROSs are produced. Accordingly, the ROS level and the ∆Ψm changes are the main indicators of mitochondrial function. Excessive ROS production results in the accumulation of dysfunctional mitochondria; the damaged mitochondrial are mainly removed by mitophagy. To investigate whether exogenous SPD could affect the integrity of mitochondrial function in cardiomyocytes under hypoxia conditions, we first examined the ROS levels using a DHE fluorescence probe (Figure 5A). We observed that the ROS production increased after cardiomyocytes were exposed to hypoxia for 24  h (P < 0.05) and that SPD inhibited the hypoxia-induced ROS production (P < 0.05); however, DFMO abolished the decrease of ROS production mediated by SPD when DFMO  were supplied to cardiomyocytes treated by SPD (P < 0.05) (Figure 5C). Furthermore, ∆Ψm was detected using TMRE  fluorescent probe (Figure 5B). The stronger the red fluorescence intensity, the higher the mitochondrial potential (P < 0.05). We noticed that the intensity of red fluorescence was significantly lower in the IUH group than in the control group (P < 0.05). Compared to the IUH  group, the red fluorescence intensity was higher in the SPD  treatment (P < 0.05), and DFMO abolished the effects of SPD once more (P < 0.05) (Figure 5D). Then we estimated the colocalization of the mitochondria (labeled with MitoTracker green) with the lysosomes (labeled by Lyso-Tracker red), which allowed us to measure the autophagic degradation of mitochondria (Figure 5E). As indicated in Figure 5F, compared to the control, hypoxia decreased the colocalization of Mito-Tracker Green and Lyso-Tracker Red in cardiomyocytes (P < 0.05). However, compared to the hypoxia group, the percentage of colocalized mitochondria and lysosomes was higher in the SPD treatment group (P < 0.05), and DFMO abolished the effect of SPD (P < 0.05). These results suggest that hypoxia increases ROS production and decreases ∆Ψm and mitophagy; SPD can alleviate hypoxia-induced mitochondrial damage by stimulating mitophagy and inhibiting oxidative stress.

4.6. Effects of SPD on myocardial MQC system in newborn off-spring exposed to IUH

Mitochondrial quality control (MQC), including the processes of mitochondrial autophagy, mitochondrial biosynthesis, and mitochondrial fusion and fission, is critical for maintaining the normal morphology, quantity,  and function of mitochondria and ensuring the normal function and metabolism of cells. Mitophagy regulates MQC and mediates extensive autophagic degradation of mitochondria in response to path-and physiological alteration. We first evaluated mitophagy by observing the formation of mitophagosome with TEM (Figure 6A - C). According to the results, some abnormal structures or fragmentation mitochondria were enclosed within a double membrane autophagic membrane, which were mitophagosomes, and indicated that mitophagy was active in the control group. However, the number of mitophagosomes highly decreased in the myocardium of the IUH  group. Interestingly, the myocardium of the SDP group showed almost identical mitophagy phenotypes to that of the control group, while DFMO abolished the effects induced by SPD. Mitochondria are highly dynamic organelles, undergoing constant fission and fusion events associated with their function. We next examined mitochondrial fragmentation using TEM (Figure 6A). We found that the numbers of small and fragment mitochondria significantly increased by IUH, compared to the control (P < 0.05), indicating that mitochondrial fission was broadly raised. This alteration was restrained by the SPD treatment,  and DFMO also abolished the role of SPD once more (Figure 6D). Furthermore, DRP1 and MFN2 (regulating proteins of mitochondrial dynamics) were detected (Figure 6E - G). MFN2 significantly decreased, whereas DRP1 increased and was observed in the IUH group compared to the control group (P < 0.05). Whereas DRP1 was down-regulated and MFN2  was upregulated with the treatment of exogenous SPD (P < 0.05), DFMO abolished the effects induced by SPD. Mitochondrial biosynthesis coordinates with mitophagy to remove damaged or excess mitochondria, generate new mitochondria, and maintain the homeostasis of MQC under stress conditions. Finally, we evaluated the expression of mitochondrial biosynthesis-related proteins, including SIRT-1, PGC-1 α, NRF-2, and TFAM, by the immunoblot analysis (Figure 6H - L). Compared to the control group, the expression of SIRT-1, PGC-1 α, NRF-2, and TFAM decreased significantly in the myocardium of the IHU group (P < 0.05). Compared to the IUH group, the protein expressions of SIRT-1, PGC-1 α, NRF-2, and TFAM in the SPD group significantly increased (P < 0.05). DFMO abolished the effect of SPD (P < 0.05). To sum up, these results indicate that IUH  leads to MQC dysfunction in the myocardial of one-day-old offspring and that the SPD treatment significantly inhibits the imbalance of the MQC system in neonatal offspring exposed to IUH.

5. Discussion 

Heart disease is a major health challenge worldwide. It has been demonstrated that chronic fetal hypoxia can trigger a fetal origin of cardiac dysfunction and program an increased risk of heart disease in the adult offspring. The heart has a high metabolic rate, and its reliance on mitochondrial function for energy production is critical for normal cardiac function. However, the effect of IUH on the cardiac mitochondrial function and its related mechanism and the focus of cardio-protection preventive strategy on mitochondria in the newborn offspring are still unknown. In the present study, we found that one-day off-spring rats exposed to IUH exhibit significant abnormalities in growth and development, cardiomyocytes maturation, especially in the myocardial mitochondrial structure and function, and exogenous SPD supplementary in utero can relieve this IUH-induced change by inhibiting myocardial oxidative stress and reducing apoptosis. Importantly,  we noticed that prenatal SPD treatment can significantly reverse the abnormalities of mitophagy and biogenesis, and mitochondrial fragmentation in the myocardium of newborn offspring rats induced by IUH. To the best of our knowledge, this research was the first report on the protective effect of SPD on IUH-induced newborn offspring’s heart damage by regulating the MQC mechanism and then improving mitochondrial function.

Sufficient evidence suggests that intrauterine growth restriction interferes with the developmental trajectory of fetal systems, which can hurt heart function later in life (5, 7, 37). In our study, IUH  caused growth retardation and heart damage in neonatal offspring rats, as the one-day newborn rats exposed to IUH exhibited a significant decrease in BW, and HW and an increase in the HW/BW ratio. Moreover, the arrangement of myocardial fibers was disordered, myofilaments were broken, inflammatory cell infiltration was noticed among myofilaments, and myocardial collagen deposition was increased. These findings are consistent with findings reported by Ducsay et al. (2) and Thompson et al. (7). In several animal models, fetal hearts exposed to chronic hypoxia exhibit reduced cardiomyocyte maturation and proliferation and decreased cardiomyocyte endowment (38, 39). Similarly, our research also showed that the proportion of binucleate cardiomyocytes increased and the expression of Ki67 decreased in the IUH group. The time at which cardiomyocytes become bi/multi-nucleated coincides with the time when mammals lose their regenerative potential (40). Fetal heart maturation is important for offspring’s heart health. Accordingly, IUH makes cardiomyocytes withdraw from the cell cycle ahead of time,  and cardiomyocyte maturation disorder leads to impaired plasticity of postnatal heart growth.

Needless to note that mitochondria are the main cellular organelles involved in the production of ROS; dysfunctional mitochondria are not only produced in response to decreased ATP production but also to increased oxidative stress and apoptosis (41). In this study, IUH significantly decreased the activity of antioxidative enzymes SOD and CAT,  decreased mitochondrial respiratory function and ATP  levels, increased the expression of BAX/BCL2, and impaired the mitochondrial structure in the myocardium of one-day-newborn offspring rats. These findings are supported by other researchers (11), who documented that maternal chronic hypoxia exposure during pregnancy could decrease the mitochondrial complex IV activity and induce apoptosis in the hearts of near-term fetal rats. We argued that mitochondrial compromise might underpin the oxidative stress and energy failure during fetal development culminating in perinatal heart injury. This can be the main reason for increased cardiovascular risk in offspring of pregnancies complicated by chronic fetal hypoxia. Accordingly, it is implementable to take mitochondrial as the target of therapeutic intervention to protect cardiac function in the offspring. Aljunaidy et al. reported that maternal treatment with mitochondrial antioxidant MitoQ prevented placental oxidative stress, rescued fetal growth,  and improved cardiac function in offspring exposed to IUH (42).

Spermidine is a polyamine with a variety of biological functions. As we mentioned, SPD can inhibit the H2O2-  induced ROS accumulation and prevent the decrease in mitochondrial membrane potential (MMP) and ATP levels in cultured neonatal rats’ cardiomyocytes and H9C2  cells. Moreover, SPD can promote mitochondrial biogenesis and improve mitochondrial function in the aging heart (43). Polyamines spermine and spermidine can affect the dynamic mitochondrial proteome, thereby preventing age-related changes in cardiac functions and cardiac aging (36). Other studies have also demonstrated that SPD supplementary delays cardiac aging by enhancing mitophagy and promoting mitochondrial respiration in mice (44). ODC is a key enzyme of polyamine synthesis, and it is reported that the in vivo knockdown of ODC  or the application of DFMO, an irreversible inhibitor of ODC results in disrupted conceptus development and fetal growth retardation due to the insufficient availability of polyamines (45, 46). Our present study indicates that treatment with 5 mg/kg/d of SPD in rats exposed to IUH  prevents growth retardation and the alteration of cardiac morphological structure and maturation in newborns off-spring. Moreover, IUH-induced cardiac oxidative stress enhancement, apoptosis increases, and mitochondrial respiratory decline in neonatal offspring were also prevented by SPD, while DFMO given to the mother abolished the SPD-mediated protection for the neonatal heart. It is well documented that polyamines involve in placental angiogenesis and embryogenesis as well as embryonic and fetal growth and development (47). Zhu et al. reported that polyamine levels were lower in the umbilical vein plasma from intrauterine growth restriction (IUGR) porcine fetuses, and supplementary SPD to the mother may prevent IUGR (48). Spermidine is a compound with reasonable safety and lower toxicity (49). The increasing numbers of preclinical and clinical studies have indicated that dietary SPD has health effects such as antiaging, cardiovascular protection, neuromodulation, and anti-inflammatory functions, such as intraperitoneal injection of SPD (50  mg/kg) in mice protects against collagen-induced arthritis by inhibiting the polarization of M1 macrophages in synovial tissue (50). Supplementation with 10 mg/kg/d of SPD reversed age-related cardiac deterioration in rats (36). More importantly, the mother was an intervention with 5 mg/kg/d of SPD during late pregnancy to improve IUHinduced cognitive and neural function decline (51) and heart oxidative stress damage in rat offspring (32). Accordingly, we used the same dose of SPD (5 mg/kg/d) for the pregnant rat exposed to IUH as in our previous study. We suspected that maternal SPD supplementation during hypoxic pregnancy might increase SPD levels in fetal circulation, which might have been related to protection against oxidative stress resulting in the placenta, which will protect the mitochondria in the developing heart from the programming by IUH, the action of SPD may be achieved by its powerful antioxidative, anti-apoptosis, and proliferation promoting mechanism.

Mitochondrial quality control mechanisms are essential for maintaining the normal morphology, number, and function of mitochondria and ensuring cells’ normal function and metabolism (16). Recent evidence has revealed that MQC mechanisms play a decisive role in perinatal myocardial mitochondrial maturation and heart development (14). Gong et al. first reported that the immature fetal-like mitochondria are directly degraded by Parkin-mediated mitophagy during the early postnatal period (19). In the meantime, mitochondrial biogenesis increases dramatically (52), and the mitochondrial fusion and fission factors are transcriptionally upregulated in the heart after birth (20, 21). However, much less is known about the effect of IUH on MCQ in the myocardium of neonatal offspring. In this study, we first observed that the number of mitophagosome decreased significantly in the myocardium of the IUH group, indicating that IUH impaired the myocardial mitophagy of newborn offspring rats. Next, we observed a significantly decreased expression of PGC-1α (a key regulator of mitochondrial biogenesis) and the expression of its upstream and downstream signaling molecules, including SIRT-1, TFAM, and NRF-2 in the myocardium of the IUH group. Moreover,  we found that the IUH exposure made smaller or fragmented mitochondria and the expression of DRP1 increased;  however, the expression of MFN2 decreased in the myocardium of newborn offspring rat, suggesting that mitochondrial fission was upregulated, and fusion was downregulated. These findings reflected the cause-and-effect relationship between the imbalance of the MQC mechanism and IUH-induced myocardium damage in newborn offspring. These complex MQC processes function in concert with one another to eliminate dysfunctional mitochondria in a specific and targeted manner and coordinate the biogenesis of new organelles, which is essential for cardiovascular homeostasis. Many intracellular and extracellular signals can regulate the MQC events, including oxidative stress, potential membrane collapse, apoptosis, and others. Here, the imbalance of mitochondrial MQC may be attributed to enhanced oxidative stress and increased apoptosis in the heart of offspring rats exposed to IUH. Accordingly, it is essential to maintain a suitable MQC mechanism to ensure a healthy mitochondrial network in the heart of newborn offspring exposed to IUH.

Given that placental SPD levels are strongly associated with fetal growth restriction (53), SPD can regulate MQC  in a different animal model. For example, SPD is a potent and specific inducer of autophagy for expanding lifespan, which can protect the aging process of several tissues, including the heart, brain, and skeletal muscle (44, 54, 55). Spermidine can alleviate cardiac aging by improving mitochondrial biogenesis (43). In this study, we applied SPD to maternal mice and found that SPD could normalize the unbalance of myocardium MQC in offspring exposed to IUH. However, maternal treatment with ODC inhibitor DFMO abolished the effects of SPD, indicating that the SPD intervention could prevent the abnormal MQC  in the myocardium of one-day offspring rats exposed to IUH. To sum up, the excellent working of MQC in the myocardium of newborn offspring could make well of mitochondrial structure, function, and maturation, thus realizing the transformation from intrauterine glycolysis capacity to postnatal fatty acid oxidation energy supply and meet the increasing heart load after birth. Furthermore,  our experiment on cardiomyocytes suggests that SPD can inhibit the increase in hypoxia-induced ROS production,  the decrease in∆Ψm, and the impaired autophagic degradation of mitochondria. We deduced that the beneficial effects of SPD could attribute to the increased SPD levels in placental and circulation by applying maternal SPD,  thereby protecting offspring myocardial mitochondrial from development programming by IUH.

5.1. Conclusion 

The present study highlights that SPD therapy in utero can protect the myocardium of one day- the offspring’s heart from the adverse effects of prenatal hypoxia. These postnatal changes caused by SPD may be achieved by antioxidation and anti-apoptosis and by regulating MQC pathways, thereby decreasing the mitochondrial development programmed in the heart exposed to IUH. There are some limitations to this study. We did not perform pharmacokinetic experiments on SPD, and thus, we cannot determine the exact SPD levels in fetal circulation when the mother was administrated with SPD. In our future studies, we will further investigate the effective blood concentration of SPD using the placenta of the fetus. The present findings provide a new framework for future studies to define the roles of SPD in preventing the myocardial mitochondria of IUH neonatal offspring.

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