Effect of Si-RNA-silenced HIF-1α gene on myocardial ischemia-reperfusion-induced insulin resistance

To explore the effect (expression and implication) of hypoxia-inducible factor-1α (HIF-1α) silence induced by siRNA on the myocardial ischemia-reperfusion-induced insulin resistance in adult rats.

One-step enzymolysis method was used to isolate adult rat cardiomyocytes; adult rat cardiomyocytes were cultured; HIF-1α gene-specific Si-RNA was constructed and transfected into rat cardiomyocytes using liposome method. Myocardial IRI model was prepared. HIF-1α and glucose transporter 4 (GLUT-4) mRNA expression was detected by RT-PCR; distribution of GLUT-4 protein expression in adult rat cardiomyocytes was detected by immunofluorescence; Western blot was used for the detection of HIF-1α protein expression; isotope tracer assay was used to detect the changes in cell glucose (Glu) uptake rate.

This method can stably get 85% to 90% active calcium tolerant adult rat cardiac myocytes, and the cultured cells were proved to be cardiomyocytes. After experiencing ischemia-reperfusion injury, HIF-1α mRNA expression levels in adult rat hypoxia cardiomyocytes had different degrees of increase compared with the control group (compared with the control group, P < 0.05). Compared with the model group, HIF-1α mRNA expression levels after ischemia and reperfusion in HIF-1αsi-RNA group and empty-vector group were lower than that in the control group and the model group; the expression reached the peak after 60 min of reperfusion, which did not change significantly in the control group. Expression of HIF-1α protein in myocardial cells was quite low in the control group; in the model group and intervention group, only after hypoxia-ischemia for 60 min, expression bands could be detected; especially in the model group, the expression had been increased until 60 min after reperfusion and began to decline from the time point of 180 min after reperfusion, but was still higher than that in the control group; in the intervention and empty-vector groups, it also increased rapidly at 60 min, but the expression was significantly lower than that in the model group; at 180 min after reperfusion, its protein expression peaked; while at 8 h after reperfusion, all the expression was extremely low. Compared with the control group, Glut4 mRNA expression in model group, transfected group and empty-vector group was reduced at the time points of T1-T4 (P < 0.05); the decline was the most significant at the time points of T1 and T2, followed by slightly increase at T3 and gradual recovery at T4; Compared with model group, Glut4 mRNA expression in transfection group was significantly reduced (P < 0.05); the decline was the most obvious at T1-T2, and then there was an increasing trend and it was recovered at T5 point. After experiencing ischemia, GLUT-4 protein expression changing trend was as follows: it was significantly reduced on the cell membrane, which was the most obvious from T1 to T3 and began to improve at T3, but still had not reached the level in the control group; it had been reached the levels of the control group at T5. After HIF-1αsi-RNA transfection and ischemia, GLUT-4 protein expression was increased in plasma and reduced on cell membrane; the decline was slightly improved at T3 and recovered to control distribution level at T5. After cardiac ischemia-reperfusion, glucose uptake rate decreased to varying degrees in myocardial cells and reached the lowest value after 60 min of ischemia, then gradually increased. After 8 h of reperfusion, the level in model group returned to the control level; compared with the model group, glucose concentration increased more serious in transfection group and empty-vector group after reperfusion.

HIF-1α played a central regulatory role in this mechanism; HIF-1α may be one of the molecular mechanisms triggering myocardial IR.