Effect And Mechanism Of Calpain On Skeletal Muscle Wasting In Severe Burned Rats

Objective: Severe burn injury causes a catabolic response that exerts profound effectson substance and energy metabolism. As the body’s largest organic nitrogen pool,skeletal muscle is the largest organ affected by catabolism. Severe burn injury can resultin immediate losses of body weight and skeletal muscle mass. These changes areassociated with the development of significant health problems, including weakness ordecreased skeletal muscle function, resulting in hypoventilation, difficulty in weaningoff respirators, decreased mobilization, or sepsis, all of which lead to significantlyincreased morbidity and mortality, and seriously affect the prognosis and quality of lifeof burnt patients. Our previous studies indicated that the main contribution to skeletalmuscle wasting after burn injury was an accelerated breakdown of myofibril proteinscaused by ubiquitin-proteasomes pathway. However, proteasome does not degradeintact myofibrils, and muscle wasting depends on an early upstream-calpian pathwaydissociation of myofibrils resulting in release of actin and myosin that are subsequentlyubiquitinated and degraded by26S proteasome. Calpains are nonlysosomal,calcium-dependent cysteine proteases and calcium is the most important activator ofcalpains. Recent studies have shown that calpain activation plays an important role inmuscle wasting caused by metabolic disease. Calpain system may be a therapeutictarget in prevention and treatment of muscle wasting. The purpose of present study is todiscuss the effects and mechanism of calpain on skeletal muscle wasting in severeburned rats. And this project was aimed to explore:(1) changes of skeletal musclewasting, skeletal muscle ultrastructure and calpain activation in skeletal muscles ofsevere scalded rats;(2) the mechanism of calpain activation on skeletal muscle wastingin severe scalded rats;(3) the reason of calpain activation in skeletal muscle caused bysevere burns;(4) the effect and mechanism of dantrolene on the skeletal muscle wastingof rats with severe scald injury.Methods: Part1. Animal experiment: Eighty male Wistar rats (180～210g) wererandomly divided into two groups: sham group (n=40) and scald group (n=40). In scaldgroup,the rats were inflicted with50%TBSA (total body surface area) full-thicknessscald by a12s immersion of back and a6s immersion of abdomen in94℃water, and anesthetized0d, ld,4d,7d or14d after injury and sacrificed for collecting arterial blood,tibialis anterior and soleus muscles. Rats and muscles were weighed and changes ofskeletal muscle ultrastructure were observed by transmission electron microscope.mRNA levels of calpain-1and calpain-2were measured by quantitative real time PCR.Levels of calpain-1and calpain-2protein were determined by Western blot, and theactivities of calpain was detected by enzyme-linked immunosorbent assay.Part2. Animal experiment: Wistar rats were randomly divided into there groups: shamgroup, scald group and calpain inhibitor group. In scald and calpain inhibitor groups,the rats were inflicted with50%TBSA full-thickness scald. After injury, the inhibitorgroup was given medication treatment by intraperitoneal injection of MDL28170(calpain inhibitor,24mg/kg), repeated every24h, and the sham and scald groupsreceived intraperitoneal injection of Ringer’s solution (2%DMSO) at the same time.Rats were anesthetized1d,4d or7d (n=8) after injury and sacrificed for collectingtibialis anterior muscles. Release of myfilaments from the myofibrils was determined bymeasuring the fraction of easily releasable myofilaments. The contents of titin, actin andmyosin in myosfilaments were measured by SDS-PAGE. The activities of calpain,26Sproteasome and caspase-3were detected by fluorescence quantitative. Apoptosis oftibialis anterior muscle cells was assessed by the assay of terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (Tunel) and the expressions ofcalpain-1, alpain-2, actin, myosin, C2subunit, protein of tBid, Cytochrome c, calpain-2in cytoplasm and mitochondria. The total and phosphorylation of Akt, mTOR, GSK-3βand FoxO3a were detected by Western blot.Cell culture experiment: L6cells were divided into3groups: control group, overexpression calpain-2group and over expression calpain-2+calpain inhibitor group. L6cell in control group was cultured with5mM CaCL2. L6cells in the over expressiongroup were received pGMLV-calpain-2-PA1virus transfection and cultured with5mMCaCL2. L6cells in the over expression+inhibitor group were receivedpGMLV-calpain-2-PA1virus transfection and cultured with5mM CaCL2+10nMMDL28170. After0h,6h and24h incubation, L6cells were collected. Cell apoptosiswas checked by AnnexinV/PI; the activitity of caspase-3was detected by fluorescencequantitative, and the expressions of tBid, Cytochrome c, calpain-2in cytoplasm andmitochondria were detected by Western blot.Part3. Animal experiment: Wistar rats were randomly divided into sham group (n=40)and scald group (n=40). Animals were anesthetized0d,1d,4d,7d or14d afterinjury and sacrificed for collecting tibialis anterior muscles. Ca2+contents of skeletalmuscle subcellular (included cytoplasm, mitochondria and sarcoplasm reticulum) weredetected by electron probe X-ray microanalysis (EPMA) way; mRNA levels of calpainactivator and calpastin were measured by quantitative real time PCR; the level ofcalpastin protein was determined by Western blot, and the activitityof calpain wasdetected by enzyme-linked immunosorbent assay (ELISA).Cell culture experiment: L6cells were divided into2group: control group andexperiment group. L6cell in control group was incubated with DMEM plus10%shamscald serum and L6cell in experiment group was cultured with DMEM containing10%scald serum for0h,6h,24h and48h. Ca2+contents in L6cell cytoplasm were detectedby Fluo4-AM and the activitity of calpain was detected by enzyme-linkedimmunosorbent assay.Part4. Animal experiment: Wistar rats were randomly divided into3groups: controlgroup (n=8), scald group (50%TBSA full-thickness scald, n=24) and dantrolenetreatment group (50%TBSA full-thickness scald, n=24). Rats in scald group receivedthe amout of diluent (5%mannitol) through caudal vein and rats in dantrolene treatmentgroup received dantrolene2mg/kg (dissolved in5%mannitol). Tibialis anterior musclesamples were harvested at Day1,4,7post scalding. Changes of skeletal muscleultrastructure were observed by transmission electron microscope, Ca2+contents ofskeletal muscle subcellular were detected by EPMA way, the levels of calpain-1andcalpain-2protein were determined by Western blot. And the activities of calpain weredetected by ELISA.Results: Part1.(1) In vivo, scald injury resulted in a rapid and persistent body weightand skeletal muscle (tibialis anterior muscle) mass loss, and tibialis anterior muscle tobody weight ratio was decreased significantly compared with sham group, especially on7d post-injury (P<0.05).(2) In scald group, disintegration or even rupture of myofibriland partial disappearance of Z lines occurred in tibialis anterior muscle after severescald injury, with mitochondrial swelling and vacuolation，and this change was basicallyconsistent with muscle tissue damage.(3) Compared with sham group, the mRNA andprotein levels of calpain-1and calpain-2in tibialis anterior muscle were increasedpost-injury, especially4d post-injury (P<0.05).(4) The activity of calpain in scald groupcontinuous increased and reached its peak after7d post-injury. Part2.(1) After severe burn injury, the fraction of easily releasable myofilaments wasincreased significantly in tibialis anterior muscle and actin, myosin and titin proteincontents were also increased remarkably. In scald group, the activity of calpainincreased and reached its peak after7d post-injury. Treatment with calpain inhinitorreduced severe burn-induced myofilaments releasing, myofibrillar proteins increasingand calpain activity.(2) Compared with the sham group, the activity of26S proteasomeand the expression of C2subunit in tibialis anterior muscle of scalded group increasedsignificantly. Treatment with calpain inhibitor MDL28170resulted in the prevention ofthe burn-induced increase in26S proteasome activity and the expression of C2subunit.(3) After injury, the apoptotic index(%) of tibialis anterior muscle was significantlyhigher accompanied by induced caspase-3activity in scalded group, especially7dpost-injury (P<0.05). Treatment with calpain inhibitor, the apoptotic index and activecaspase-3were all significantly lower, when compared with the burn group. Theexpression of calpain-2in cytoplasm and mitochondria of scalded and calpain inhibitorgroups were all increased, but the calpain-2level of inhibitor group was lower than thescalded group. The expression of Bid in cytoplasm had no significant difference duringthe three groups, whereas the expression of tBid in mitochondria of scalded group washigher than that of sham and inhibitor groups post-injury, especially1d and7dpost-injury. Low-level expression of cytochrome c protein was in cytoplasm, whereasmost cytocrome c protein expressed in mitochondria. In scalded group, most cytocromec protein expressed in cytoplasm and the content of cytocrome c was sharply decreased7d post-injury. In vitro, the cell apoptosis rate and caspase-3activity of over-expressioncalpain group were markedly elevated, higher than that of control and inhibitor groups.With the increment of incubation time, the expressions of calpain-2and tBid wereincreased in over-expression group’s L6cell mitochondria, significantly difference withcontrol and inhibitor groups. The expressions of cytocrome c protein were gradual risein cytoplasm and gradual reduce in mitochondria in over-expression and inhibitor group,but the difference between the two groups was statistical significantly (P<0.05).(4) Thephosphorylated level of Akt and its downstream targets mTOR, GSK-3β and FoxO3a intibialis anterior muscle significantly decreased after severe scald injury and thephosphorylated level of Akt, mTOR and GSK-3β significally incereased when calpaininhibitor MDL28170was given by intraperitoneal injection, especially1d and7d afterinjury. Part3.(1) The content of Ca2+in tibialis anterior muscle’s cytoplasm and mitochondriarise while the content of Ca2+in muslce’s sarcoplasmic reduced in scaled group,especially1d post-injury. The mRNA and protein levels of calpastatin decreased both at1d and4d post-injury, followed by an increase from7d in scald group, which hadsignificantly difference with sham group. The mRNA level of calpain activator didn’tchange significantly after injury.(2) Scald rat serum caused increasing of cytoplasmCa2+content in L6cell. And with the extended period, the content of cytoplasm Ca2+and the calpain activation increased in L6cell.Part4.(1) The ryanodine receptor antagonist dantrolene significant reduced the overloado calcium in tibialis anterior muscle cell’s cytoplasm and mitochondria. The contents ofCa2+at cytoplasm and mitochondria were significantlylower in dantrolene group thanthose in scald group at1and4days after injury.(2) There were no significantultrastructure changes in dantrolene treatment group after injury and caplain-1andcalpain-2protein levels in dantrolene treatmentobviously lower than those in scaldgroup respectively.(3) The activities of calpain in dantrolene treatment group had noobviously change compared with that in control group, and dantrolene protected fromsekeletal muscle mass and weight loss induced by severe scalded injury.Conclusions:(1) Activation of the calpain system is the major cause of severe burn-induced muscle wasting. The mechanism of calpain activaton regulating muscle massare calpain disruption of myofibril with release of actin and myosin, and probably othermyofilaments, as an integral part of protein degradation; calpain activates theubiquitin-proteasome pathway by increasing substrate flux and26S proteasomeactivation; calpain reactivates the mitochondrial pathways of apoptosis and promote cellapoptosis; calpain activation downregulates Akt and its downstream signaling molecules,accompanied with inhibiting protein synthesis and promoting muscle proteindegradation.(2) Muscle calcium uptake is increased and intracellular overload inskeletal muscle after severe burn injury. And this is the reason for calpain activation andmuscle proteolysis after burn injury.(3)The ryanodine receptor antagonist dantroleneoffers significant protection from sekeletal muscle tissue damage and minimizes tibialisanterior muscle ultrastructural change induced by severe scalded injury.