Hypoxia/reoxygenation (H/R) inhibits proliferation and induces apoptosis in H9C2 cell.
To investigate the effect of H/R on H9C2 cell, we first analyzed the transcriptomic signature of GSE103731 and 112 differentially expressed genes (DEGs) were identified (Fig. 1A), with multiple DEGs were enriched in the mitosis-related pathways (Fig. 1B). After exposure of H9C2 cells to hypoxic conditions followed by reoxygenation, the proliferation capacity was detected (Fig. 1C). As shown in Fig. 1D, the viability of H9C2 was significantly attenuated with the dependence of time after H/R. The flow cytometry analysis of Ki67, the proliferation marker that expresses in the nuclei of proliferating cells, was also decreased in H9C2 cell after H/R (Fig. 1E), indicating a downregulation of proliferation in H9C2 cells post H/R treatment. Additionally, we performed GSEA analysis of GSE103731 and noticed that the pathway of apoptosis was upregulated in H/R-treated H9C2 (Fig. 1F). Meanwhile, apoptosis of H9C2 after H/R was also promoted (Fig. 1G). These results collectively suggest that H/R contributes to MIRI by inhibiting cell proliferation and enhancing apoptosis.
H/R induced apoptosis through aggravating ferroptosis by targeting NRF2/system Xc-/GPX4 axis in H9C2 cell.
To investigate the mechanism of H/R-induced apoptosis in H9C2, we first performed GO-GSEA analysis in H9C2 cell after H/R derived from GSE103731 and the results showed that glutamate/glutathione metabolic processes were downregulated (Fig. 2A), which have been reported be involved in ferroptosis. In contrast, lipid-related metabolic processes including lipid oxidation, the characteristics of ferroptosis, were upregulated (Fig. 2A), suggesting that H/R might confer apoptosis by inducing ferroptosis in H9C2 cell. Since ferroptosis is an iron-depended cell death driven by Fe2+, we then detected the content of Fe2+ in each group of H9C2 cell using iron assay kit. As shown in Fig. 2B, Fe2+ significantly increased H9C2 cell after H/R treatment. Fe2+ can generate superfluous ROS via the Fenton reaction[8], we then determined the ROS level and found that ROS level significantly increased in H/R-treated H9C2 cell (Fig. 2C). Consistently, lipid peroxidation, as judged by lipid ROS (Fig. 2D) and MDA (Fig. 2E) content were also increased. These results indicated that H/R was sufficient to induce ferroptosis in H9C2 cell.
To further investigate the specific molecular mechanisms of H/R-induced ferroptosis in H9C2 cell, we first detected the expression of ferroptosis-related protein. As shown in Fig. 2F, NRF2, xCT, SLC3A2, GPX4 and FTH1 in H/R group were significantly decreased. Since the GSH level was regulated by cystine imported by xCT[14], we detected the GSH content and noticed that GSH was decreased in H/R-treated H9C2 cell (Fig. 2G). Meanwhile, NADPH is an intracellular agent that reduces the conversion between GSH and GSSG, leading to a reduction of lipid peroxides[15]. Correspondingly, the ratio of NADP+/NADPH was increased in H/R-treated H9C2 cell (Fig. 2H). We therefore preliminarily conclude that H/R regulates the Nrf2/System Xc-/GPX4 axis to promote ferroptosis. To investigate whether H/R exerts apoptosis through ferroptosis, we exposed H/R-treated H9C2 cell to an ferroptosis inhibitor. As shown in Fig. 2I, DFO and ferrostain-1 (fer-1), the inhibitor of ferroptosis, restored the reduced expression of GPX4 and FTH1. Meanwhile, lipid ROX was decreased by DFO and fer-1 (Fig. 2J), which indicated that ferroptosis of H9C2 cell induced by H/R was attenuated by DFO and fer-1. Consistently, apoptosis of H9C2 cell driven by H/R was rescued by DFO and fer-1 (Fig. 2K). These results revealed that H/R induced apoptosis through aggravating ferroptosis by targeting NRF2/system Xc-/GPX4 axis and accumulating iron in H9C2 cell.
H/R aggravated ferroptosis through lactate production.
Previous study reported that iron metabolism can be modulated by lactate in mice[16]. To assess whether lactate participated in ferroptosis induced by H/R in H9C2 cell. H9C2 cell was first cultured in hypoxia followed by culturing in high- or low-glucose culture medium to modulate lactate production (Figure S1A). Compared to the high-glucose culture medium, intracellular and extracellular lactate levels both decreased in the low-glucose group (Figure S1B, S1C). Meanwhile, iron content was also reduced by low glucose, suggesting that iron content was positively related with lactate in H/R-treated H9C2 cell (Fig. 3A). Since iron can generate ROS through Fenton reaction, we next detected the ROS in different groups. As shown in Fig. 3B, ROS level decreased in the low-glucose group (Fig. 3B). Consistently, the markers of ferroptosis including MDA and lipid ROS reduced when cultured in low-glucose culture medium (Fig. 3C,3D). These results preliminary revealed that ferroptosis could be regulated by lactate. To further confirm that lactate induced ferroptosis, the H9C2 cell was precultured in glucose-free culture medium for 6 hours to remove intracellular lactate. The cells were then cultured under hypoxia followed by reoxygenation with different concentrations of lactate. Lipid peroxidation including MDA (Fig. 3E) and lipid ROS (Fig. 3F) levels changed with lactate concentration. Conversely, fer-1 rescued the lipid peroxidation induced by lactate (Fig. 3G, S1D), indicating that lactate could positively regulate ferroptosis in H/R-induced ferroptosis in H9C2 cell.
Lactate induced ferroptosis via induction of SLC39A14 expression.
To understand how lactate induced ferroptosis, we treated H9C2 cell with different concentrations of lactate followed by detection of ferroptosis-related genes expression. Of note, SLC39a14, a transmembrane transporter that modulated the cellular uptake of manganese, iron and zinc, was upregulated by lactate with the lactate content (Fig. 4A). Likewise, iron content was significantly promoted after exposing H9C2 cell to lactate (Fig. 4B). We then detected iron uptake by addition of extra iron in the culture medium followed by culturing H9C2 cell with lactate. The result showed that iron uptake and the content of intracellular iron increased in lactate-treated H9C2 cells (Fig. 4C, S2A), suggesting that SLC39A14 might be responsible for elevated iron induced by lactate. To further confirm the regulatory role of SLC39A14 in iron uptake and ferroptosis, we constructed an SLC39A14 knockdown H9C2 cell (Fig. 4D) followed by iron content detection. As expected, iron was decreased after SLC39A14 knockdown (Fig. 4E). encouragingly, iron uptake and intracellular iron were also reduced in SLC39A14 knock-out cell (Fig. 4F, S2B). In addition, SLC39A14 expression was downregulated after exposing H9C2 cell to 2-deoxy-d-glucose (Fig. 4G). Moreover, lipid ROS level significantly decreased after SLC39A14 knocking down (Fig. 4H). These results indicated that SLC39A14 contributed to lactate-induced ferroptosis in H9C2 cell. Since apoptosis was induced by ferroptosis in H9C2 cell (Fig. 2K), we therefore measured the apoptosis rate of each group of H9C2. As presented in Fig. 4I, apoptosis was positively correlated with lactate exposure with lactate content dependence. However, knock-out of SLC39A14 could significantly rescue apoptosis induced by ferroptosis (Fig. 4J). Together, these results showed that lactate induced ferroptosis in H9C2 cell required the expression of SLC39A14.
Serum lactate correlates with iron content and myocardial injury markers in humans.
To confirm the regulatory role of lactate in myocardial injury, we surveyed arterial blood from 40 patients with myocardial and evaluated the correlation between various laboratory index. (Table S1). It is reported that the concentration of serum lactate content ranges from 0.5-2mM, and our sample from myocardial injury patients varied from 0.4 to 4.2mM. Glucose was found to be positively correlated with lactate in serum, as lactate is a product of glycolysis. Additionally, a positive correlation was observed between lactate, creatine kinase (CK), and CK myocardial band isoenzyme (CK-MB), which are biomarkers of myocardial damage (Fig. 5B, 5C). Meanwhile, ejection fraction (EF) has been reported to be associated with poor outcomes in myocardial injury[17], we therefore evaluated the EF and found that lactate reduced the EF in myocardial injury patients (Fig. 5D, S3). These results indicated that a high concentration of lactate could aggravate myocardium injury.
To understand whether lactate aggravated myocardium injury through elevating Fe2+ uptake followed by ferroptosis, we examined serum Fe2+ from 40 patients. Consistent with the previous finding which lactate induced Fe2+ uptake. Serum Fe2+ was negatively correlated with serum glucose and lactate (Fig. 5E, 5F). In addition, we also observed a negative correlation between serum Fe2+, CK and CK-MB (Fig. 5G, 5H), while a positive correlation was found between serum Fe2+ and EF (Fig. 5I). These data further demonstrated that lactate induced Fe2+ uptake followed by ferroptosis, which led to myocardium injury ultimately.