GPX3 Overexpression Ameliorates Cardiac Injury Post Myocardial Infarction Through Activating LSD1/Hif1α Axis

FIGURE 1. The expression of Glutathione peroxidase 3 (GPX3) was significantly upregulated after acute myocardial infarction (AMI). (A) The volcano plot displays the significant‐down, significant‐up and nonsignificant proteins in normal and ischaemic swine heart tissues. Proteins with fold changes greater than two‐fold and a p value less than 0.05 are included. (B) Spatial expression of GPX3 in human heart samples with acute myocardial infarction. (C) Boxplot depicting the expression levels of GPX3 in human cardiomyocytes following MI, categorised into control, ischaemic and border zone (IZ + BZ) and remote zone (RZ). Data obtained from single‐nucleus RNA sequencing (snRNA‐seq). (D) Boxplot depicting the expression levels of GPX3 in mouse cardiomyocytes at 1, 7 and 14 days post‐AMI. Data are categorised into control, ischaemic and border zone (IZ + BZ) and remote zone (RZ) from single‐nucleus RNA sequencing (snRNA‐seq). (E) Measurement of GPX3 activity levels in the plasma of patients with or without AMI within 7 days, using a colorimetric assay kit (n = 40). (F) Quantitative real‐time PCR (qRT‐PCR) analysis of GPX3 mRNA levels in cardiac tissues from mice subjected to MI or sham surgery,…

FIGURE 2. Effects of GPX3 (Glutathione peroxidase 3) on oxidative stress and apoptosis in NRCMs. (A) Representative confocal microscope images and quantitative analysis of reactive oxygen species (ROS) levels after DHE staining in neonatal rat cardiomyocytes (NRCMs) with GPX3 overexpression or knockdown (n = 3). (B) Representative confocal microscope images and quantitative analysis of mitochondrial ROS using MitoSOX Red staining in NRCMs across four experimental groups: Control, OGD, OGD + GPX3 and OGD + GPX3i (n = 3). (C) JC‐1 staining and quantitative analysis to detect changes in mitochondrial membrane potential (MMP) in CMs treated differently (n = 3). (D) Western blot analysis showing the protein levels of Bcl‐2, Bax, Cleaved‐caspase‐3 and GPX3 in CMs under various treatments (n = 3). (E) Flow cytometry analysis of caspase‐3 activity in CMs (n = 3). (F) Flow cytometry assessment of early and late apoptotic cells in CMs (n = 3). (G) TUNEL staining of CMs subjected to OGD conditions (n = 3). Data are presented as mean ± SEM. Significance levels are indicated as follows: *p < 0.05, **p < 0.01, ***p < 0.001.

FIGURE 3. GPX3 (Glutathione peroxidase 3) overexpression reduced cardiomyocyte loss and preserved cardiac function in mice 28 days post‐MI in vivo. (A) Experimental protocol: Adenovirus 5 (Ad5) was injected into the myocardium at the infarct border immediately after ligation of the left anterior descending artery. Apoptosis rates and relative protein levels were assessed on day 3 post‐MI. Echocardiography was performed on day 28 post‐infarction to evaluate cardiac function, followed by the harvesting of hearts for further analysis. (B) Western blot analysis of Bcl‐2, Bax, Cleaved‐caspase‐3 and GPX3 protein levels in mouse hearts (n = 3). (C) Representative images and quantitative data of TUNEL staining among different groups (n = 4). (D–E) Echocardiographic measurements of left ventricular ejection fraction (LVEF) and left ventricular fractional shortening (LVFS) among the different groups (n = 6). (F) Representative images and quantification of Masson's trichrome and Sirius red staining at 28 days following MI or sham surgery (n = 5). (G) Protein levels and quantitative analysis of Collagen I, MMP2 and α‐SMA in heart tissue post‐surgery (n = 5). Data are presented as mean ± SEM.…

FIGURE 4. Glutathione peroxidase 3 (GPX3) overexpression promotes Hif1α to mitigate apoptosis. (A) Western blot analysis of Hif1α and GPX3 protein levels in CMs with GPX3 overexpression or knockdown, measured under hypoxic conditions at various time points (n = 3). (B) Hif1α mRNA levels in CMs assessed under normoxic conditions following various treatments (n = 3). (C) Hif1α mRNA levels in CMs measured under hypoxic conditions after different treatments (n = 3). (D) Western blot analysis of Hif1α, Bcl‐2 and Bax protein levels in OGD‐induced CMs transfected with either Ad5:cTNT‐GPX3, Ad5:cTNT‐Hif1αi or both (n = 3). Data are presented as mean ± SEM. Significance levels are indicated as follows: *p < 0.05, **p < 0.01, ***p < 0.001.

FIGURE 5. Glutathione peroxidase 3 (GPX3) overexpression enhances Hif1α expression via activation of LSD1. (A) A heatmap displaying protein variations in cardiomyocytes treated with GPX3 or a control. (B, C) The mRNA levels of LSD1 and GPX3 in control, GPX3 overexpression and GPX3 inhibition groups (n = 3). (D) Protein levels and quantification of Hif1α, LSD1 and GPX3 in CMs with GPX3 overexpression or knockdown under normoxic conditions (n = 3). (E) Protein levels and quantification of Hif1α, LSD1 and GPX3 in CMs with GPX3 overexpression or knockdown under hypoxic conditions (n = 3). (F) Protein levels and quantification of LSD1, Hif1α, Bcl‐2 and Bax in OGD‐induced CMs with or without LSD1 overexpression (n = 3). (G) Protein levels and quantification of LSD1, Hif1α, Bcl‐2 and Bax in OGD‐induced NRCMs with or without LSD1 knockdown (n = 3). Data are presented as mean ± SEM. Significance levels are indicated as follows: *p < 0.05, **p < 0.01, ***p < 0.001.

FIGURE 6. Glutathione peroxidase 3 (GPX3) attenuated the loss of cardiomyocytes via the GPX3/LSD1/Hif1α pathway. (A) Protein quantification and levels of LSD1, Hif1α, Bcl‐2, Bax and Cleaved‐caspase‐3 in CMs subjected to OGD and transfected with Ad5:cTNT‐GPX3, Ad5:cTNT‐LSD1i or both (n = 3). (B) Representative confocal microscope images and quantitative analysis of JC‐1 assays detecting MMP in CMs treated with OGD, OGD + GPX3, OGD + LSD1i and OGD + GPX3 + LSD1i (n = 3). (C, D) Representative images and quantitative analysis of TUNEL staining in NRCMs subjected to OGD (n = 3) and in mouse hearts three days post‐MI (n = 4) following various interventions. Data are presented as mean ± SEM. Significance levels are indicated as follows: *p < 0.05, **p < 0.01, ***p < 0.001.

FIGURE 7. Summary of GPX3 (Glutathione peroxidase 3) on cardiac MI injury. In myocardial tissues subjected to hypoxic conditions following a myocardial infarction, GPX3 expression is significantly upregulated. This increase in GPX3 is associated with a reduction in oxidative stress and apoptosis, which collectively contribute to improved cardiac function. The protective role of GPX3 is partly mediated through its interaction within the GPX3/LSD1/Hif1α axis, enhancing the cellular resilience to hypoxic stress.