| Ischemic postconditioning (I-postC) is an amenable protective strategy to protect tissue against ischemia-reperfusion (I/R) injury and can be evoked by cycles of brief intermittent interruption of blood flow to tissues at the immediate onset of reperfusion after a prolonged period of ischemia. I-postC has 2 distinct phases of protection:one is the early phase or first window of protection, which appears immediately after I-postC stimulus and lasts 2 h or so; and the other is the delayed phase or second window of protection, which reappears 12-24 h after I-postC insult. The protective effect of I-postC on the myocardium has been mimicked in vitro by hypoxic postconditioning (H-postC).Although several studies reported that I-postC reduces skeletal muscle injury induced by I/R, the mechanism by which I-postC suppresses skeletal muscle injury remains largely unknown.Numerous studies found that intracellular Ca2+ signaling is involved in mediating I/R injury. Calcineurin (CaN), a Ca2+/calmodulin-dependent protein phosphatase, plays a pivotal role in I/R injury of many cell types. We previously reported on CaN involved in I-postC-induced cardioprotection. Therefore, we hypothesized that I-postC attenuates I/R injury through CaN signaling pathway in skeletal muscle. We aimed to investigate the effect of I-postC or H-postC on skeletal muscle injury induced by I/R or hypoxia-reoxygenation (H/R) through a CaN pathway in rat skeletal muscle or skeletal muscle cells (SMCs).1 Ischemic postconditioning protects skeletal muscle from ischemia/reperfusion injuryThe present study was aimed to investigate the effect of I-postC or H-postC on I/R or H/R injury. I/R injury in the right hind limb of healthy male Wistar rats was induced by clamping the right femoral artery. Rats were randomly divided into 4 groups:(1) I/R group:right femoral artery occluded for 4 h then reperfused for 2 h (I/R-2h),12 h (I/R-12h) or 24 h (I/R-24h) (n=8 at each time point). (2) I-postC group: after 4 h of right femoral artery occlusion, I-postC was initiated for 3 cycles of 1-min reperfusion and 1-min ischemia and then samples collected at 2,12 or 24 h after reperfusion (n=8 at each time point). (3) ischemic preconditioning (IPC) group:the right femoral arteries of rats was occluded for 5 min followed by 5 min of reperfusion, repeated for three cycles before I/R. (4) Sham group:rats underwent the same surgical procedure without clamping (n=8 at each time point). Hemodynamic, wet/dry weight ratio (W/D), malondialdehyde (MDA) levels, lactate dehydrogenase (LDH) concentration in blood plasma, and ultra microstructure of skeletal muscle were detected. Cultured skeletal muscle cells from neonatal Wistar rat were divided into 4 groups:H/R group, H-postC group, hypoxic preconditioning (HPC) group and control group. H/R was produced by 2-hour hypoxia/24-hour reoxygenation. The survival rate and apoptotic rate of skeletal muscle cells in each group were measured. Western blot was used to detect the expressions of CRT. CaN and glucose-regulated protein 78 (GRP 78). The results were as follows:During in vivo experiment, compared with I/R. I-postC significantly decreased LDH concentration. MDA levels and W/D. attenuated the ultramicrostructure injury of skeletal muscle and the apoptosis of nucleolus.24 h after reperfusion, compared with that in I/R group, the expression of CRT, CaN and GRP 78 in I-postC group increased significantly. In cultured skeletal muscle cells, H-postC attenuated cell injury induced by H/R. Compared with those in H/R group. CRT and CaN expressions in H-postC increased significantly. 2 Ischemic postconditioning protects skeletal muscle from ischemia/ reperfusion injury through improving microcirculation in rat hind limbsTo investigate the effects of I-postC on skeletal muscle microcirculation and I/R injury in rat hind limbs. Rats were randomly divided into 4 groups:I/R group, I-postC group, IPC group, Sham group. Microcirculation were detected on 2h,12h and 24h after reperfusion. It was found that blood velocity in venule increased in I-postC group compared with that in I/R group and internal diameter of arteriole and venule increased compared with those in I/R groupsignificantly.3 Calcineurin mediates the protective effect of ischemic postconditioning on skeletal muscle from ischemia/reperfusion injuryThe present study was aimed to investigate whether CaN was involved in intracellular signal transduction both in vivo and in cultured skeletal muscle cells.Exposure of skeletal muscles to I-postC greatly increased the protein level of CaN (P<0.05 versus I/R), along with decreased MDA level and LDH concentration in rat plasma. Furthermore, we postconditioned cultured SMCs and analyzed the protein level of CaN and cell death. CaN protein level was upregulated by postconditioning in cultured cells, which was accompanied by an increase in cell survival and decrease in apoptosis rate. CaN may be involved in the protective role of I-postC on skeletal muscle from I/R injury. To analyze this suggestion, we treated rats and cultured SMCs with CsA, a CaN inhibitor. CsA decreased the expression and activity of CaN in skeletal muscles of I-postC rats and significantly elevated the ratio of W/D and MDA level and LDH concentration in plasma. In postconditioned SMCs, CsA reduced the expression and activity of CaN, and cell survival was significantly reduced and apoptosis elevated. Furthermore, overexpression of CaN in SMCs significantly elevated cell survival and greatly reduced apoptosis under H-postC. Thus, CaN may be required for the protective effect of I-postC on skeletal muscle injury.The conclusions were as follows:Firstly, I-postC attenuates skeletal muscle I/R injury. Secondly, I-postC can protect the skeletal muscle from reperfusion injury through improving microcirculation. Finally, I-postC can protect skeletal muscle in rat against I/R injury through CaN activation. These data shed light on the complex signaling cascades involved in the cytoprotection of postconditioning on skeletal muscles. |
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