3.1 SpostC upregulates HIF-1α signaling pathway in healthy myocardium and enhances BNIP3-mediated mitochondrial autophagy.
Oxidative stress in myocardial tissue: The level of myocardial oxidative stress was determined after the application of HIF-1α specific blocker 2ME2. At the end of 2 hours of reperfusion, serum LDH, CKM, CK-MB, TNNT2, and IL-6 levels were significantly increased in the C-I/R and C-SpostC+2ME2 groups compared with the C-sham group (P<0.05). And compared with the C-I/R group, serum LDH, CKM, CK-MB, TNNT2, and IL-6 levels changed significantly in the C-SpostC and C-I/R+2ME2 groups (P<0.05), confirming that 2ME2 abrogated the above effects of SpostC (P<0.05). (Table1, Fig. 1)
Echocardiographic assessment: Compared with the C-sham group, the C-I/R group had significantly lower stroke volume (SV)%, ejection fraction (EF)%, and fraction shortening (FS)% due to dysfunction of the left ventricular wall (P<0.05).In addition, the diastolic left ventricular diameter (LVIDd) and systolic left ventricular diameter (LVIDs) in C-I / R group were significantly increased. At the same time, the end-diastolic left ventricular anterior wall thickness (LVAWd), systolic left ventricular anterior wall thickness (LVAWs), diastolic left ventricular posterior wall thickness (LVPWd), Systolic left ventricular posterior wall thickness (LVPWs) were significantly decreased (P<0.05). SpostC could improve the above cardiac function indexes, making it significantly better than the C-I/R group, but the HIF-1α blocker completely abrogated the ability of SpostC to improve cardiac function (P<0.05). There was no significant difference in cardiac function indexes between the C-I/R and C-SpostC+2ME2 groups (P>0.05). (Table2, Fig. 2)
HE staining of cardiomyocytes: Microscopic observation showed that the myocardial tissue was more regularly arranged in the C-sham group, with only focal disorganization, a small number of cardiomyocytes were swollen, and myocardial fibers were fractured; myocardial tissue was arranged irregularly in the C-I/R group, cardiomyocytes were swollen and vacuolar degeneration, some myocardial fibers were fractured, and a small number of chronic inflammatory cells infiltrated; compared with the C-I/R group, the disorder of myocardial arrangement, cardiomyocytes swelling, myocardial fiber fracture and inflammatory cell infiltration in the C-SpostC group were alleviated. There was no significant difference in pathological changes between the C-SpostC+2ME2 group and the C-I/R group, but the pathological changes were more severe in the C-SpostC + 2ME2 group. (Fig. 3)
Myocardial mitochondrial ultrastructure: Myocardial mitochondrial ultrastructure was observed by TEM. C-sham group: myocardial cell nucleus, myocardial fiber, and mitochondrial structure are intact. C-I/R group: myocardial structure was severely damaged, myofibrils were basically aligned, and myotome was slightly shortened segmentally. Mitochondria were obviously swollen, and mitochondrial cristae were extensively dissolved into flocculent, outer membrane segmentally dissolved, matrix slightly dissolved, visible autophagosomes, more lipid droplets. C-SpostC group: myocardial structure was clear, myofibrils were basically aligned, myotome length was uniform; mitochondrial cristae were extensively dissolved into flocculent, outer membrane segmentally dissolved, autophagosomes were visible. C-SpostC+2ME2 group: myocardial structure was damaged, matrix dissolved; mitochondria were obviously swollen, similar to C-I/R group. (Fig. 4)
Changes in HIF-1α subunit and hypoxia-sensitive gene expression: HIF-1α subunit and hypoxia-sensitive genes are crucial in resisting myocardial I/R injury and regulating mitochondrial function. It was found that SpostC was able to upregulate HIF-1α and downstream LC3-II, BNIP3, Beclin1, and TLR9 levels after I/R injury (P<0.05, Table2-1, Table2-2, Fig. 2), but the use of 2ME2 abrogated the above effects of SpostC.(P<0.05). (Table3, 4, Fig. 5)
Myocardial apoptosis levels: When I/R injury occurred in healthy myocardium, the apoptosis rate increased, and the survival rate decreased. SPostC reduced the apoptosis rate compared with the C-I/R group (P<0.05). C-SpostC+2ME2 significantly increased the apoptosis rate after I/R injury compared with the C-SPostC group (C-SPostC group: 20.881±5.477% vs C-SpostC+2ME2 group: 44.566±7.179%, P<0.05) (Fig. 6)
Myocardial infarct size: SpostC significantly reduced myocardial infarct size after I/R injury in healthy myocardium. Infarct size (IS)/ischemic myocardial area (Area at risk, AAR) (19.37±2.97% in C-SpostC group vs. 49.56±1.77% in C-I/R group, P<0.05), but the myocardial infarct size increased to 53.23±2.84% after application of 2ME2. (Fig. 7)
3.2 Impaired HIF-1α in Aged Myocardium Leads to diminished BNIP3-Mediated Mitochondrial Autophagy, but SpostC Still Plays an Excellent Myocardial Protective Role
Oxidative stress in myocardial tissue: At the end of 2 hours of reperfusion, serum LDH, CKM, CK-MB, TNNT2, and IL-6 levels were significantly increased in the L-I/R and L-SpostC+2ME2 groups compared with the L-sham group (P<0.05). And SpostC significantly decreased serum LDH, CKM, CK-MB, TNNT2, and IL-6 levels compared with the L-I/R group (P<0.05). (Table5, Fig. 8)
Echocardiographic assessment: Compared with the L-sham group, the L-I/R group had significantly lower stroke volume (SV)%, EF%, and FS% due to left ventricular ischemia-reperfusion injury (P<0.05). In addition, LVIDd and LVIDs in the L-I/R group were significantly increased, whereas LVAWd, LVAWs, LVPWd, and LVPWs were significantly decreased (P<0.05). SpostC could improve the above cardiac function indexes in aged heart, making it significantly better than those in the L-I/R group, but the ability of SpostC to improve cardiac function was completely abrogated by the HIF-1α blocker 2ME2 (P<0.05). there was no significant difference in cardiac function indexes between the L-I/R and L-SpostC+2ME2 groups (P>0.05). (Table6, Fig. 9)
HE staining of cardiomyocytes: Microscopic observation showed that the structure of myocardial tissue was basically normal in L-sham group, with only focal cardiomyocyte swelling and a small amount of chronic inflammatory cell infiltration; myocardial tissue was arranged irregularly in the L-I/R group, cardiomyocytes were swollen and vacuolar degeneration, some myocardial fibers were fractured, more chronic inflammatory cells were infiltrated, and local necrosis was observed. Compared with the L-I/R group, the disorder of myocardial arrangement, myocardial cell swelling, myocardial fiber fracture, and inflammatory cell infiltration were reduced in the L-SpostC group. Compared with the L-I/R group, the pathological changes were more severe in the L-SpostC+2ME2 group. ( Fig. 10 )
Myocardial mitochondrial ultrastructure: L-sham group: myofibrils were basically aligned, myotome length was basically uniform; mitochondrial cristae were extensively dissolved into flocculent, outer membrane segmentally dissolved, matrix segmentally dissolved. L-I/R group: mitochondria were obviously swollen, and mitochondrial cristae were extensively dissolved into flocculent, outer membrane segmentally dissolved, matrix obviously dissolved, autophagosomes were visible. However, the mitochondrial morphology of the L-SpostC group was still good, only some mitochondria were slightly swollen, and no noticeable dissolution of myofilament was observed. The damage of myocardial ultrastructure in L-SpostC+2ME2 group was similar to that in L-I/R group. (Fig. 11)
Changes in HIF-1α subunit and hypoxia-sensitive gene expression: We further determined the expression of HIF-1α and downstream LC3-II, BNIP3, Beclin1, TLR9, and IL-6 proteins. The results showed that the expression of HIF-1α and downstream LC3-II, BNIP3, Beclin1, and TLR9 was significantly increased in the L-SpostC group, whereas IL -6 protein expression was significantly decreased (P<0.05 compared with L-I/R and L-SpostC+2ME2 groups). However, the protective effect of the L-SpostC+2ME2 group on the above proteins was completely reversed after the administration of 2ME2, in which LC3-II protein levels were not significantly different in the L-I/R and L-SpostC+2ME2 groups (P > 0.05). (Table7, 8, Fig. 12)
Myocardial apoptosis levels: In the aged myocardium, SPostC reduced the apoptosis rate after I/R injury from 40.05+5.51% to 25.52+8.50% (P<0.05). In contrast, the apoptosis rate after I/R injury was significantly increased in the L-SpostC+2ME2 group after the use of HIF-1α blocker 2ME2 (L-SpostC group: 25.52+8.50 vs. L-SpostC+2ME2 group: 53.98+7.37, P< 0.05). (Fig. 13)
Myocardial infarction size: We used 2ME2 to block HIF-1α signaling pathway in the aged myocardium and measured infarct size changes. SpostC significantly reduced myocardial infarct size in aged myocardium after I/R. (31.06±3.20% in C-SPostC group vs. 57.54±3.50% in C-I/R group, P<0.05). After administration of 2ME2, myocardial infarct size was significantly increased in the L-SpostC+2ME2 group (59.63±5.71%, P<0.05 compared with the L-I/R+SpostC group), confirming that the myocardial protective effect of SpostC was abrogated after the use of HIF-1α-specific blocker. (Fig. 14)