| Acute myocardial ischemia is a common cardiovascular emergency, but also an important cause of death in patients with coronary arterial disease. However, even the blood flow is restored with effective treatments such as thrombolytic therapy, coronary artery bypass surgery and percutaneous coronary intervention, the cardiac function can not recovered completely. This suggested that some irreversible structural damages appeared in myocardium after coronary artery ischemia.Some evidences indicate that the cytoskeleton injury first comes into irreversible damage in ischemic myocardium.30 min and 90 min after myocardial ischemia, actin and vinculin will reduce significantly respectively. Our previous studies have also found that the decrease of actin, desmin, tubulin and other cytoskeletal proteins during endotoxemia was one of the important causes which can reduce the systolic and diastolic function of myocardium. Thus, cytoskeletal proteins play an important role in the regulation of myocardial function.F-actin is not only a kind of cytoskeleton but also a contractile protein in myocardium, which is located in myocardial membrane and Z band with a variety of actin binding proteins. As one of the binding proteins, a-actinin can conduct mechanical force between sarcomere by anchored the opposite ends of the actin filaments at a central Z-body or Z-line. Thus, whenα-actinin is removed, the force may not be effectively transmitted at the ends of the cell, and it will directly affect the systolic and diastolic function of myocardium. Therefore, this study was focued on the relationship between expression ofα-actinin and myocardial dysfunction after acute coronary artery ischemia. This study was divided into following two parts.In the first part,32 rats were randomly divided into 4 groups:sham, simple ischemia for 30 minutes (I30min), simple ischemia for 1 hour (I1h), ischemia for 1 hour and then reperfusion for 2 hours (IR). In order to determine whether ischemia can destroyα-actinin, we observed the expression ofα-actinin by immunohistochemistry assay. We also measure the expression of PLC and PI3K and observe the relationship between changes ofα-actinin and cardiac function during myocardial ischemia.In the second part, adult rat cardiomyocytes were isolated and cultured in M199 medium. The rat cardiomyocytes were pretreated with PLC inhibitor (LY-29400) 10μg/ml or PI3K inhibitor (U-73122) 1μg/ml. Then we observed the changes of a-actinin and F-actin and determine the role of PLC and PI3K on cardiomyocytes under hypoxic ischemic condition and further explore the effects ofα-actinin and on cardiac function during hypoxic ischemia.The results were as follows:In the first part:1. The changes of cardiac function during acute left anterior descending coronary artery ischemia and reperfusion in rats.The LVSP was significantly different among the different groups (F= 12.788 P=0.000). LVSP decreased with prolonged ischemia time. After reperfusion, LVSP partly recovered, but was still lower than the sham group (P=0.001). Compared to the sham group, the LVSP decreased significantly in I30min, I60min and IR group (P values were 0.001,0.000 and 0.001). Compared to the I60min group, it increased significantly in I30min and IR group compared with the I60min (P values were 0.033 and 0.037).The LVEDP was significantly different among the four groups (F= 14.965, P=0.000). It gradually increased with the increase of ischemia time and partly recovered after reperfusion, but was still higher than the sham group. The highest value of LVEDP was in I60min group. Compared to the sham group, the LVEDP increased significantly in I30min, I60min and IR group (All of the P values were 0.000). And it was significantly different between I30min group and I60min group (P=0.037).The +dp/dt max was changed significantly among the four groups (F=16.532, P=0.000). It decreased gradually with prolonged ischemia time and partly recovered after reperfusion, but was still lower than the sham group. The lowest value of +dp/dt max was in I60min group. Compared to the sham group, the+dp/dt max reduced significantly in I30min, I60min and IR group (All of the P values were 0.000).The -dp/dt max was also changed among the four groups (F=8.995, P=0.000). It gradually decreased with the increase of ischemia time and partly recovered after reperfusion, but was still lower than the sham group. The lowest value of-dp/dt max was in I60min group. The -dp/dt max in I30min, I60min and IR group was reduced significantly compared with the sham group (P values were 0.002,0.000 and 0.001).2. The pathological analysis of ischemic myocardium:There were no significant pathological changes in myocardial tissue in sham group:regular myocardial fibers, normal morphology, integrity structure and clear transverse striation. While in I30min group, myocardial fibers became loose arrangement and there were RBC in myocardial interstitium. In I60min group, part of the myocardial fibers arranged in waves, sarcoplasm eosinophilic weakened, interstitial edema occured, fibers fractured and nuclear freed. In IR group, fractured myocardial fibers were still visible and there were also vacuoles under the membrane.3. The immunohistochemical analysis of a-actinin:The positive expression of a-actinin was brown or tan in myocardial fibers which mainly arranged along the cardiac Z lines. In sham group, a-actinin distributed and stained regularly. With prolonged ischemia time, the distribution of a-actinin became uneven with punctiform or lamellar absence. While in IR group, the a-actinin was also uneven, but the gray value was higher than the ischemic groups. The gray level of a-actinin was significant different among the four groups (F=12.802, P=0.000). The lowest level of a-actinin was in I60min group (121.31±5.89), and it reduced significantly compared to sham group (P=0.000).4. The content of PLC PI3K and in myocardium:The level of PI3K was significantly different among the four groups (F=10.976, P=0.000). It gradually increased with the increase of the ischemia time. Compared to the sham group, the level of PI3K in I60min group increased significantly (P=0.000). And it was also significant higher than that in the I30min group (P=0.042).The content of PLC was significantly different among the four groups (F=7.275, P=0.001). The level of PLC gradually increased with the prolonged ischemia time and partly recovered after reperfusion. Compared to the sham group, the level of PLC in I60min group increased significantly (P=0.001). And it was also significant higher than that in the I30min group (P=0.002).In the second part:1. The content of LDH in culture medium:The activity of LDH in culture medium was significantly different among the five groups (F=10.892, P=0.000). LDH content was lower in the control group and it was significantly increased in I60min, IR, LY-294002 and U-73122 group compared to the control group (All of the P values were 0.000).2. The survival rate of cardiomyoctye:The survival rate of cardiomyoctye was significantly different among the five groups (F=42.618,P=0.000). The survival rate was highest in the control group, it reduced in I60min group and it was much lower in LY-294002 and U-73122 group. Compared with the control group, the survival rates in LY-294002 and U-73122 group were decreased significantly (Both of the P values were 0.000) and they were also significantly different from that in I60min(P values were 0.016 and 0.002).3. The systolic and diastolic function of single cardiomyoctye:The contraction amplitude of single cardiomyoctye was significantly different among the five groups (F=15.893, P=0.000). In control group, it was largest. In I60min group, it reduced significantly compared to the control group (P=0.000). While, data analysis displaied the differences between I60min group and LY-294002 group (P= 0.000) and between I60min group and U-73122 group (P=0.001). And it did not change in LY-294002 group compared with the control group (P=0.689).4. The fluorescence intensity ofα-actininThe fluorescence intensity ofα-actinin was significantly different among the five groups (F=28.770, P=0.000). It was highest in control group and lowest in I60min group and the difference between the two groups was significant (P=0.001). While after LY-294002 and U-73122 treatment, it increased significantly compared to that in I60min group (Both of the P values were 0.000), but was still lower than that in control group (P values were 0.000 and 0.001).5. The content of Ca2+-Mg2+-ATPaseThe content of Ca2+-Mg2+-ATPase was significantly different among the five groups (F=14.668,P=0.000). It was highest in control group and lowest in I60min group and the difference between the two groups was significant (P=0.000). After LY-294002 and U-73122 treatment, it increased significantly compared to that in I60min group (Both of the P values were 0.000), but was still lower than that in control group (P values were 0.033 and 0.020).Conclusions:1.60 min of left anterior descending coronary artery ischemia can cause myocardial dysfunction and cytoskeletal structural damage in vivo.2. Cardiomyocardial cytoskeletal protein a-actinin changes may be one of the reasons which caused cardiac dysfunction during left anterior descending coronary artery ischemia.3. Cardiomyocardial cytoskeletal protein a-actinin changes during left anterior descending coronary artery ischemia may be related to intracellular signaling pathways PI3K and PLC activation. |
PDF Full Text Request