Noninvasive remote ischemic preconditioning for global protection of skeletal muscle against infarction

The aim of this study was to investigate the efficacy and mechanism of action of a noninvasive remote ischemic preconditioning (IPC) technique for the protection of multiple distant skeletal muscles against ischemic necrosis (infarction). It was observed in the pig that three cycles of 10-min occlusion and reperfusion in a hindlimb by tourniquet application reduced the infarction of latissimus dorsi (LD), gracilis (GC), and rectus abdominis (RA) muscle flaps by 55%, 60%, and 55%, respectively, compared with their corresponding control (n = 6, P < 0.01) when they were subsequently subjected to 4 h of ischemia and 48 h of reperfusion. This infarct-protective effect of remote IPC in LD muscle flaps was abolished by an intravenous bolus injection of the nonselective opioid receptor antagonist naloxone (3 mg/kg) 10 min before remote IPC and a continuous intravenous infusion (3 mg/kg) during remote IPC and by an intravenous bolus injection of the selective delta 1-opioid receptor antagonist 7-benzylidenealtrexone maleate (3 mg/kg). However, this infarct-protective effect of remote IPC was not affected by an intravenous bolus injection of the ganglionic blocker hexamethonium chloride (20 mg/kg) or the nonspecific adenosine receptor antagonist 8-(p-sulfophenyl)theophylline (10 mg/kg) or by a local intra-arterial injection of the adenosine1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (3 mg/muscle flap) given 10 min before remote IPC. It was also observed that this remote IPC of skeletal muscle against infarction was associated with a slower rate of muscle ATP depletion during the 4 h of sustained ischemia and a reduced muscle neutrophilic myeloperoxidase activity after 1.5 h of reperfusion. These observations led us to speculate that noninvasive remote IPC by brief cycles of occlusion and reperfusion in a pig hindlimb is effective in global protection of skeletal muscle against infarction. This infarct-protective effect is most likely triggered by the activation of opioid receptors in the skeletal muscle, and remote IPC is associated with an energy-sparing effect during sustained ischemia and attenuation of neutrophil accumulation during reperfusion.     

Mitochondrial KATP channels in hindlimb remote ischemic preconditioning of skeletal muscle against infarction

We previously demonstrated in the pig that instigation of three cycles of 10 min of occlusion and reperfusion in a hindlimb by tourniquet application (approximately 300 mmHg) elicited protection against ischemia-reperfusion injury (infarction) in multiple distant skeletal muscles subsequently subjected to 4 h of ischemia and 48 h of reperfusion, but the mechanism was not studied. The aim of this project was to test our hypothesis that mitochondrial ATP-sensitive potassium (KATP) (mKATP) channels play a central role in the trigger and mediator mechanisms of hindlimb remote ischemic preconditioning (IPC) of skeletal muscle against infarction in the pig. We observed in the pig that hindlimb remote IPC reduced the infarct size of latissimus dorsi (LD) muscle flaps (8 x 13 cm) from 45 +/- 2% to 22 +/- 3% (n = 10; P < 0.05). The nonselective KATP channel inhibitor glibenclamide (0.3 mg/kg) or the selective mKATP channel inhibitor 5-hydroxydecanoate (5-HD, 5 mg/kg), but not the selective sarcolemmal KATP (sKATP) channel inhibitor HMR-1098 (3 mg/kg), abolished the infarct-protective effect of hindlimb remote IPC in LD muscle flaps (n = 10, P < 0.05) when these drugs were injected intravenously at 10 min before remote IPC. In addition, intravenous bolus injection of glibenclamide (1 mg/kg) or 5-HD (10 mg/kg) at the end of hindlimb remote IPC also abolished the infarct protection in LD muscle flaps (n = 10; P < 0.05). Furthermore, intravenous injection of the specific mKATPchannel opener BMS-191095 (2 mg/kg) at 10 min before 4 h of ischemia protected the LD muscle flap against infarction to a similar extent as hindlimb remote IPC, and this infarct-protective effect of BMS-191095 was abolished by intravenous bolus injection of 5-HD (5 mg/kg) at 10 min before or after intravenous injection of BMS-191095 (n = 10; P < 0.05). The infarct protective effect of BMS-191095 was associated with a higher muscle content of ATP at the end of 4 h of ischemia and a decrease in muscle neutrophilic myeloperoxidase activity at the end of 1.5 h of reperfusion compared with the time-matched control (n = 10, P < 0.05). These observations led us to conclude that mKATP channels play a central role in the trigger and mediator mechanisms of hindlimb remote IPC of skeletal muscle against infarction in the pig, and the opening of mKATP channels in ischemic skeletal muscle is associated with an ATP-sparing effect during sustained ischemia and attenuation of neutrophil accumulation during reperfusion.     

Early and late effects of ischemic preconditioning on microcirculation of skeletal muscle flaps

The present study was designed to investigate the early and late effects of ischemic preconditioning on muscle flap perfusion and reperfusion-induced skeletal muscle damage. Thirty-six Sprague-Dawley rats were divided into six experimental groups of six animals each. The cremaster muscle flap model and the intravital microscopy system were used to observe microcirculatory changes associated with ischemia-reperfusion injury and ischemic preconditioning. In groups 1, 2, and 3, microcirculatory measurements were taken on the same day; however, in groups 4, 5, and 6, measurements were taken a day after surgery. Group 1 served as a control. The cremaster muscle was prepared as a tube flap, subjected to an hour of perfusion without ischemia. In group 2 (ischemic preconditioning + ischemia group), the cremaster muscle tube flap was subjected to 30 minutes of ischemia and 30 minutes of reperfusion, followed by 4 hours of total ischemia. In group 3 (ischemia alone), the flap was submitted to 4 hours of ischemia alone. In group 4 (control), the cremaster muscle flaps were dissected out, preserved in the subcutaneous tunnel, and submitted to 24 hours of perfusion only. In group 5 (ischemic preconditioning + 24 hours of perfusion + 4 hours of ischemia), the ischemic preconditioning protocol was followed by 24 hours of perfusion and 4 hours of ischemia. In group 6 (24 hours of perfusion + ischemia), the same protocol was used as in group 5 without ischemic preconditioning. Functional capillary perfusion, and the diameters of the arterioles of the first, second, and third order were significantly increased in the ischemic preconditioning group during the early period, but not after 24 hours of perfusion. No differences in the red blood cell velocities of arterioles of the first, second, or third order were found in either the early-effect or late-effect groups. The numbers of rolling, adhering, and transmigrating leukocytes, however, were significantly lower in the ischemic preconditioning group at both early and late follow-up. Ischemic preconditioning of the skeletal muscle flap has both an early and a late protective effect against reperfusion injury. Ischemic preconditioning at the early interval significantly improves muscle flow hemodynamics of the flap and attenuates leukocyte-mediated reperfusion injury. After 24 hours of reperfusion, however, ischemic preconditioning failed to improve the flow hemodynamics of the flap, yet it still protected the skeletal muscle flap from leukocyte-mediated reperfusion injury.     

缓激肽在骨骼肌缺血预适应对猪心肌坏死和凋亡中的作用

目的：研究缓激肽在骨骼肌缺血预适应对心肌坏死和心肌凋亡保护中可能的作用：方法：采用非开胸法建立猪心脏缺血／再灌注（I／R）模型,通过球囊堵塞左股动脉造成骨骼肌短暂缺血,使用缓激肽（BK）的B2受体拮抗剂烟酸已可碱（HOE-140）以及外源BK进行干预。分别观察各组对心肌坏死和凋亡的影响。结果：远端预处理后心肌坏死范围明显缩小,凋亡率明显降低：预处理前使用HOE-140可使对坏死范围的保护作用明显减弱;心肌I／R前使用外源BK注射,可缩小心肌梗死范围。但HOE-140及外源BK对以上凋亡指标无影响．结论：骨骼肌远端预适应可减少心肌坏死和心肌凋亡、BK可能参与对坏死面积的保护．但不参与对凋亡的保护作用。     

Role and mechanism of PKC in ischemic preconditioning of pig skeletal muscle against infarction

Protein kinase C (PKC) inhibitors, chelerythrine (Chel, 0.6 mg) and polymyxin B (Poly B, 1.0 mg), and PKC activators, phorbol 12-myristate 13-acetate (PMA, 0.05 mg) and 1-oleoyl-2-acetyl glycerol (OAG, 0.1 mg), were used as probes to investigate the role of PKC in mediation of ischemic preconditioning (IPC) of noncontracting pig latissimus dorsi (LD) muscles against infarction in vivo. These drugs were delivered to each LD muscle flap (8 x 12 cm) by 10 min of local intra-arterial infusion. It was observed that LD muscle flaps sustained 43 +/- 5% infarction when subjected to 4 h of global ischemia and 24 h of reperfusion. IPC with three cycles of 10 min ischemia-reperfusion reduced muscle infarction to 25 +/- 3% (P < 0.05). This anti-infarction effect of IPC was blocked by Chel (42 +/- 7%) and Poly B (37 +/- 2%) and mimicked by PMA (19 +/- 10%) and OAG (14 +/- 5%) treatments (P < 0.05), given 10 min before 4 h of ischemia. In addition, the ATP-sensitive K(+) (K(ATP)) channel antagonist sodium 5-hydroxydecanoate attenuated (P < 0.05) the anti-infarction effect of IPC (37 +/- 2%), PMA (44 +/- 17%), and OAG (46 +/- 9%). IPC, OAG, and Chel treatment alone did not affect mean arterial blood pressure or muscle blood flow assessed by 15-microm radioactive microspheres. Western blot analysis of muscle biopsies obtained before (baseline) and after IPC demonstrated seven cytosol-associated isoforms, with nPKCepsilon alone demonstrating progressive cytosol-to-membrane translocation within 10 min after the final ischemia period of IPC. Using differential fractionation, it was observed that nPKCepsilon translocated to a membrane compartment other than the sarcolemma and/or sarcoplasmic reticulum. Furthermore, IPC and preischemic OAG but not postischemic OAG treatment reduced (P < 0.05) muscle myeloperoxidase activity compared with time-matched ischemic controls during 16 h of reperfusion after 4 h of ischemia. Taken together, these observations indicate that PKC plays a central role in the anti-infarction effect of IPC in pig LD muscles, most likely through a PKC-K(ATP) channel-linked signal-transduction pathway.     

骨骼肌缺血预适应对猪心肌凋亡的影响及阿片受体的作用

目的:确定骨骼肌缺血预适应对猪心肌凋亡及其调控基因Bcl-2/Bax的影响,并探讨阿片受体在此机制中可能的作用。方法:采用非开胸法建立猪心脏缺血/再灌注(I/R)模型,通过球囊堵塞左股动脉造成骨骼肌短暂缺血,分别使用阿片受体拮抗剂纳洛酮以及神经节阻断剂六烃己胺进行干预。采用末端探针标记及流式细胞技术检测心肌凋亡细胞及调控基因Bcl-2/Bax,确定各组对以上指标的影响。结果:①和缺血对照组(CONT组)相比,远端预处理后心肌细胞凋亡率明显降低(4.43%±0.74%vs15.4%±1.15%,P<0.05),提示骨骼肌远端预适应(RP)可减少心肌凋亡。②和CONT组相比,远端预处理后Bcl-2/Bax比值明显增高(1.36±0.09vs0.56±0.08,P<0.05),提示RP对心肌凋亡的影响可能通过影响Bcl-2/Bax进行调控。③预处理前使用阿片受体拮抗剂纳洛酮可使以上保护作用减弱(P<0.05),但纳洛酮对CONT组无影响。④预处理前使用神经节阻断剂六烃己胺,不影响RP对心肌的保护作用。结论:骨骼肌缺血预适应减少心肌细胞凋亡,可能通过影响Bcl-2/Bax进行调控;阿片受体可能参与此保护作用,且不通过神经反射进行。     

Postconditioning for salvage of ischemic skeletal muscle from reperfusion injury: efficacy and mechanism

We tested our hypothesis that postischemic conditioning (PostC) is effective in salvage of ischemic skeletal muscle from reperfusion injury and the mechanism involves inhibition of opening of the mitochondrial permeability transition pore (mPTP). In bilateral 8x13 cm pig latissimus dorsi muscle flaps subjected to 4 h ischemia, muscle infarction increased from 22+/-4 to 41+/-1% between 2 and 24 h reperfusion and remained unchanged at 48 (38+/-6%) and 72 (40+/-1%) h reperfusion (P<0.05; n=4 pigs). PostC induced by four cycles of 30-s reperfusion/reocclusion at the onset of reperfusion after 4 h ischemia reduced muscle infarction from 44+/-2 to 22+/-2% at 48 h reperfusion. This infarct protective effect of PostC was mimicked by intravenous injection of the mPTP opening inhibitor cyclosporin A or NIM-811 (10 mg/kg) at 5 min before the end of 4 h ischemia and was abolished by intravenous injection of the mPTP opener atractyloside (10 mg/kg) at 5 min before PostC (P<0.05; n=4-5 pigs). PostC or intravenous cyclosporin A injection at 5 min before reperfusion caused a decrease in muscle myeloperoxidase activity and mitochondrial free Ca2+ concentration and an increase in muscle ATP content after 4 h ischemia and 2 h reperfusion compared with the time-matched controls. These effects of PostC were abolished by intravenous injection of atractyloside at 5 min before PostC (P<0.05; n=6 pigs). These observations support our hypothesis that PostC is effective in salvage of ischemic skeletal muscle from reperfusion injury and the mechanism involves inhibition of opening of the mPTP.     

Signal mechanism activated by erythropoietin preconditioning and remote renal preconditioning-induced cardioprotection

It has been recently reported that release of erythropoietin could mediate the cardioprotective effects of remote renal preconditioning. However, the mechanism of erythropoietin-mediated cardioprotection in remote preconditioning is still unexplored. Therefore, the present study was designed to investigate the possible signal transduction pathway of erythropoietin-mediated cardioprotection in remote preconditioning in rats. Remote renal preconditioning was performed by four episodes of 5 min renal artery occlusion followed by 5 min reperfusion. Isolated rat hearts were perfused on Langendorff apparatus and were subjected to global ischemia for 30 min followed by 120 min reperfusion. The levels of lactate dehydrogenase (LDH) and creatine kinase (CK) were measured in coronary effluent to assess the degree of myocardial injury. Extent of myocardial infarct size and coronary flow rate was also measured. Remote renal preconditioning and erythropoietin preconditioning (5,000 IUkg(-1), i.p.) attenuated ischemia-reperfusion-induced myocardial injury and produced cardioprotective effects. However, administration of diethyldithiocarbamic acid (150 mg kg(-1) i.p.), a selective NFkB inhibitor, and glibenclamide (5 mg kg(-1) i.p.), a selective K(ATP) channel blocker, attenuated cardioprotective effects of remote preconditioning and erythropoietin preconditioning. However, administration of minoxidil (1 mg kg(-1) i.v.), a selective K(ATP) channel opener, restored the attenuated cardioprotective effects of remote preconditioning and erythropoietin preconditioning in diethyldithiocarbamic acid pretreated rats. These results suggest that K(ATP) channel is a downstream mediator of NFkB activation in remote preconditioning and erythropoietin preconditioning. Therefore, it may be concluded that erythropoietin preconditioning and remote renal preconditioning trigger similar signaling mechanisms for cardioprotection, i.e., NFkB activation followed by opening of K(ATP) channels.     

外泌体在远隔缺血预适应中对神经细胞的保护作用

缺血性卒中会对大脑神经细胞产生损伤,远隔缺血预适应(remote ischemic preconditioning,RIP)作为一种内源性的保护机制,通过远端器官或组织缺血/再灌注而产生的保护性物质经体液运输作用于神经中枢,发挥对神经细胞的保护作用,有望被应用于卒中的临床治疗。越来越多的研究发现,RIP过程中分泌的外泌体极可能是其产生的保护性物质,本文通过对外泌体在RIP中对神经细胞的保护作用进行综述,为探索远隔缺血预适应对神经元的保护机制提供新思路。     

Local and remote arteriolar dilations initiated by skeletal muscle contraction

To investigate the relationship between skeletal muscle metabolism and arteriolar dilations in the region local to contracting muscle fibers as well as dilations at remote arteriolar regions upstream, we used a microelectrode on cremaster muscle of anesthetized hamsters to stimulate four to five muscle fibers lying approximately perpendicular to and overlapping a transverse arteriole. Before, during, and after muscle contraction, we measured the diameter of the arteriole at the site of muscle fiber overlap (local) and at a remote site approximately 1,000 microm upstream. Two minutes of 2-, 4-, or 8-Hz stimulation (5-10 V, 0.4-ms duration) produced a significant dilation locally (8.2 +/- 2.0-, 22.5 +/- 2.4-, and 30.9 +/- 2.1-microm increase, respectively) and at the remote site (4.2 +/- 0.8, 11.0 +/- 1.1, and 18.9 +/- 2.7 microm, respectively). Muscle contraction at 4 Hz initiated a remote dilation that was unaffected by 15-min micropipette application of either 2 microM tetrodotoxin, 0.07% halothane, or 40 microM 18-beta-glycyrrhetinic acid between the local and upstream site. Therefore, at the arteriolar level, muscle contraction initiates a robust remote dilation that does not appear to be transmitted via perivascular nerves or gap junctions.     

Reduction of skeletal muscle injury in composite tissue allotransplantation by heat stress preconditioning

Ischemia-reperfusion injury is a dominant factor limiting tissue survival in any microsurgical tissue transplantation, a fact that also applies to allogeneic hand transplantation. The clinical experience of the 12 human hand transplantations indicates that shorter ischemia times result in reduced tissue damage and, ultimately, in better hand function. Heat stress preconditioning and the accompanying up-regulation of the heat shock protein 72 have been shown to reduce the ischemia-reperfusion injury following ischemia of various organs, including organ transplantation. The aim of this study was to reduce the ischemia-reperfusion injury in a model of composite tissue allotransplantation. Allogeneic hind limb transplantations were performed from Lewis (donor) to Brown-Norway rats. Donor rats in group A (n = 10) received a prior heat shock whereas rats in group B (n = 10) did not receive any prior heat shock. Group C served as a control group without transplantation. The transplantations were performed 24 hours after the heat shock, at which time the heat shock protein 72 was shown to be up-regulated. The outcome was evaluated 24 hours after transplantation by nitroblue tetrazolium staining and wet-to-dry weight ratio of muscle slices (anterior tibial muscle). The nitroblue tetrazolium staining showed a significant reduction of necrotic muscle in group A (prior heat shock) (p = 0.005). The wet-to-dry ratio was significantly reduced in group A (prior heat shock), indicating less muscle edema and less tissue damage (p = 0.05). Heat shock preconditioning 24 hours before an ischemic event leads to an up-regulation of heat shock protein 72 in muscle and to a tissue protection reducing ischemia-reperfusion injury in composite tissue transplantation.     

NO modulates norepinephrine release in human skeletal muscle: implications for neural preconditioning

The purpose of this study was to estimate muscle interstitial norepinephrine (NE) levels during exercise and to determine whether nitric oxide (NO) modulates NE release in the skeletal muscle in humans. We measured interstitial dialysate concentrations of NE with two microdialysis probes inserted into the forearm. Probes were perfused with saline and the NO synthesis inhibitor N(G)-monomethyl-L-arginine (L-NMMA), respectively. Dialysate samples were collected during two sequential 20-min intense dynamic handgrip periods, preceded by 40-min baseline periods. On a different day, forearm ischemia was performed instead of the first exercise period. Exercise increased dialysate NE from 172 +/- 42 to 270 +/- 45 pg/ml (83% increase, P < 0.02, n = 6). Probes perfused with L-NMMA had a 136 +/- 39% greater dialysate NE compared with probes perfused with saline (225 +/- 25 vs. 125 +/- 25 pg/ml, P < 0.001, n = 9). The exercise-induced increase in NE (125 +/- 52%) was attenuated if preceded by exercise (34 +/- 34%) or ischemia (40 +/- 36%; P = 0.06, n = 6), suggesting a neural preconditioning effect. This attenuation was not observed in probes perfused with L-NMMA. We propose that NO modulates NE release in skeletal muscle, that ischemic exercise increases muscle interstitial NE, and that this increase can be attenuated by a preconditioning effect mediated in part by NO.     

The cardioprotection of the late phase of ischemic preconditioning is enhanced by postconditioning via a COX-2-mediated mechanism in conscious rats

The present study sought to determine whether the combination of late preconditioning (PC) with postconditioning enhances the reduction in infarct size. Chronically instrumented rats were assigned to a 45-min (subset 1) or 60-min (subset 2) coronary occlusion followed by 24 h of reperfusion. In each subset, rats received no further intervention (control) or were preconditioned 24 h before occlusion (PC), postconditioned at the onset of reperfusion following occlusion, or preconditioned and postconditioned without (PC + postconditioning) or with the COX-2 inhibitor celecoxib (3 mg/kg ip; PC + postconditioning + celecoxib) 10 min before postconditioning. Myocardial cyclooxygenase-2 (COX-2) protein expression and COX-2 activity (assessed as myocardial levels of PGE(2)) were measured 6 min after reperfusion in an additional five groups (control, PC, postconditioning, PC + postconditioning, and PC + postconditioning + celecoxib) subjected to a 45-min occlusion. PC alone reduced infarct size after a 45-min occlusion but not after a 60-min occlusion. Postconditioning alone did not reduce infarct size in either setting. However, the combination of late PC and postconditioning resulted in a robust infarct-sparing effect in both settings, suggesting additive cardioprotection. Celecoxib completely abrogated the infarct-sparing effect of the combined interventions in both settings. Late PC increased COX-2 protein expression and PGE(2) content. PGE(2) content (but not COX-2 protein) was further increased by the combination of both interventions, suggesting that postconditioning increases the activity of COX-2 induced by late PC. In conclusion, the combination of late PC and postconditioning produces additive protection, likely due to a postconditioning-induced enhancement of COX-2 activity.     

KATP channels of mouse skeletal muscle: mechanism of channel blockage by AMP-PNP

Single ATP-sensitive potassium channels (K _ATP channels) were studied in inside-out membrane patches excised from mouse skeletal muscle. Channel blockage by the non-hydrolysable ATP analogue AMP-PNP was investigated in the absence or presence of 1 mM MgCl₂ with K⁺-rich solutions bathing the internal membrane surface. Currents through single. K _ATP channels were recorded at –40 and +40 mV AMP-PNP (5 to 500 M; Li salt) reduced the open-probability p_o of K _ATP channels and decreased the single-channel currents at high nucleotide concentrations by approximately 10%. Half maximal reduction of p_o at –40 mV was observed at nucleotide concentrations of 29 M in the absence and of 39 M in the presence of Mg²⁺. The steepness of the AMP-PNP concentration-response curves was strongly affected by Mg²⁺, the Hill coefficients of the curves were 0.6 in the absence and 1.6 in the presence of 1 mM MgCl₂. The efficacies of channel blockage by AMP-PNP at –40 and ⁺40 mV were not significantly different. The results indicate that a K _ATP channel can bind more divalent Mg²⁺-complexes of AMP-PNP than trivalent protonated forms of the nucleotide and that channel blockage is hardly affected by the membrane electric field. To estimate the contribution of lithium ions to the observed results, we studied the effects of LiCl (0.8 to 10 mM) in the Mg²⁺-free solution on the single channel current i. At a Li⁺ concentration of 10 mM, i was hardly affected at –40 mV but reduced by a factor of 0.75 at +40 mV. The results are interpreted by a fast, voltage-dependent blockage of K _ATP channels by internal Li⁺ ions. Correspondence to: B. Neumcke     

Electromechanical coupling between skeletal and cardiac muscle. Implications for infarct repair

Skeletal myoblasts form grafts of mature muscle in injured hearts, and these grafts contract when exogenously stimulated. It is not known, however, whether cardiac muscle can form electromechanical junctions with skeletal muscle and induce its synchronous contraction. Here, we report that undifferentiated rat skeletal myoblasts expressed N-cadherin and connexin43, major adhesion and gap junction proteins of the intercalated disk, yet both proteins were markedly downregulated after differentiation into myo-tubes. Similarly, differentiated skeletal muscle grafts in injured hearts had no detectable N-cadherin or connexin43; hence, electromechanical coupling did not occur after in vivo grafting. In contrast, when neonatal or adult cardiomyocytes were cocultured with skeletal muscle, approximately 10% of the skeletal myotubes contracted in synchrony with adjacent cardiomyocytes. Isoproterenol increased myotube contraction rates by 25% in coculture without affecting myotubes in monoculture, indicating the cardiomyocytes were the pacemakers. The gap junction inhibitor heptanol aborted myotube contractions but left spontaneous contractions of individual cardiomyocytes intact, suggesting myotubes were activated via gap junctions. Confocal microscopy revealed the expression of cadherin and connexin43 at junctions between myotubes and neonatal or adult cardiomyocytes in vitro. After microinjection, myotubes transferred dye to neonatal cardiomyocytes via gap junctions. Calcium imaging revealed synchronous calcium transients in cardiomyocytes and myotubes. Thus, cardiomyocytes can form electromechanical junctions with some skeletal myotubes in coculture and induce their synchronous contraction via gap junctions. Although the mechanism remains to be determined, if similar junctions could be induced in vivo, they might be sufficient to make skeletal muscle grafts beat synchronously with host myocardium.     

Development of an in vitro model for study of the efficacy of ischemic preconditioning in human skeletal muscle against ischemia-reperfusion injury.

Ischemia-reperfusion (I/R) injury causes skeletal muscle infarction and ischemic preconditioning (IPC) augments ischemic tolerance in animal models. To date, this has not been demonstrated in human skeletal muscle. This study aimed to develop an in vitro model to investigate the efficacy of simulated IPC in human skeletal muscle. Human skeletal muscle strips were equilibrated in oxygenated Krebs-Henseleit-HEPES buffer (37 degrees C). Aerobic and reperfusion phases were simulated by normoxic incubation and reoxygenation, respectively. Ischemia was simulated by hypoxic incubation. Energy store, cell viability, and cellular injury were assessed using ATP, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT), and lactate dehydrogenase (LDH) assays, respectively. Morphological integrity was assessed using electron microscopy. Studies were designed to test stability of the preparation (n = 5-11) under normoxic incubation over 24 h; the effect of 1, 2, 3, 4, or 6 h hypoxia followed by 2 h of reoxygenation; and the protective effect of hypoxic preconditioning (HPC; 5 min of hypoxia/5 min of reoxygenation) before 3 h of hypoxia/2 h of reoxygenation. Over 24 h of normoxic incubation, muscle strips remained physiologically intact as assessed by MTT, ATP, and LDH assays. After 3 h of hypoxia/2 h of reoxygenation, MTT reduction levels declined to 50.1 +/- 5.5% (P < 0.05). MTT reduction levels in HPC (82.3 +/- 10.8%) and normoxic control (81.3 +/- 10.2%) groups were similar and higher (P < 0.05) than the 3 h of hypoxia/2 h of reoxygenation group (45.2 +/- 5.8%). Ultrastructural morphology was preserved in normoxic and HPC groups but not in the hypoxia/reoxygenation group. This is the first study to characterize a stable in vitro model of human skeletal muscle and to demonstrate a protective effect of HPC in human skeletal muscle against hypoxia/reoxygenation-induced injury.     

Coordination of skeletal muscle gene expression occurs late in mammalian development

The acquisition of specialized skeletal muscle fiber phenotypes during development is investigated by systematic measurement of the accumulation of 21 contractile protein mRNAs during hindlimb development in the rat and the human. During early myotube formation in both species there is no coordination of expression of either fast or slow contractile protein isoform genes, but rather some slow, some fast, and some cardiac isoforms are expressed. Some isoforms are not detected at all in early myotubes. From Embryonic Day 19 in the rat, and after 14 weeks in the human, a strong bias toward fast isoform expression is evident for all gene families examined. This results in the establishment of a coordinated fast isoform phenotype at birth in the rat, and by 24 weeks in the human fetus. Unexpectedly, during secondary myotube formation in the rat we observe sudden rises and falls in contractile protein gene output. We interpret these fluctuations in terms of periods of myoblast proliferation followed by synchronized fusion into myotubes. The data presented indicate that each contractile protein gene has its own determinants of mRNA accumulation and that the different myoblast populations which contribute to the developing limb are not intrinsically programmed to produce particular coordinated phenotypes with respect to the non-myosin heavy chain contractile proteins.     

Sepsis stimulates release of myofilaments in skeletal muscle by a calcium-dependent mechanism.

Sepsis is associated with a pronounced catabolic response in skeletal muscle, mainly reflecting degradation of the myofibrillar proteins actin and myosin. Recent studies suggest that sepsis-induced muscle proteolysis may reflect ubiquitin-proteasome-dependent protein breakdown. An apparently conflicting observation is that the ubiquitin-proteasome pathway does not degrade intact myofibrils. Thus, it is possible that actin and myosin need to be released from the myofibrils before they can be ubiquitinated and degraded by the proteasome. We tested the hypothesis that sepsis results in disruption of Z-bands, increased expression of calpains, and calcium-dependent release of myofilaments in skeletal muscle. Sepsis induced in rats by cecal ligation and puncture resulted in increased gene expression of micro-calpain, m-calpain, and p94 and in Z-band disintegration in the extensor digitorum longus muscle. The release of myofilaments from myofibrillar proteins was increased in septic muscle. This response to sepsis was blocked by treating the rats with dantrolene, a substance that inhibits the release of calcium from intracellular stores to the cytoplasm. The present results provide evidence that sepsis is associated with Z-band disintegration and a calcium-dependent release of myofilaments in skeletal muscle. Release of myofilaments may be an initial and perhaps rate-limiting component of sepsis-induced muscle breakdown.