Barium chloride injures myofibers through calcium-induced proteolysis with fragmentation of motor nerves and microvessels

Background Local injection of BaCl2 is an established model of acute injury to study the regeneration of skeletal muscle. However, the mechanism by which BaCl2 causes muscle injury is unresolved. Because Ba2+ inhibits K+ channels, we hypothesized that BaCl2 induces myofiber depolarization leading to Ca2+ overload, proteolysis, and membrane disruption. While BaCl2 spares resident satellite cells, its effect on other tissue components integral to contractile function has not been defined. We therefore asked whether motor nerves and microvessels, which control and supply myofibers, are injured by BaCl2 treatment. Methods The intact extensor digitorum longus (EDL) muscle was isolated from male mice (aged 3–4 months) and irrigated with physiological salt solution (PSS) at 37 °C. Myofiber membrane potential (Vm) was recorded using sharp microelectrodes while intracellular calcium concentration ([Ca2+]i) was evaluated with Fura 2 dye. Isometric force production of EDL was measured in situ, proteolytic activity was quantified by calpain degradation of αII-spectrin, and membrane disruption was marked by nuclear staining with propidium iodide (PI). To test for effects on motor nerves and microvessels, tibialis anterior or gluteus maximus muscles were injected with 1.2% BaCl2 (50–75 μL) in vivo followed by immunostaining to evaluate the integrity of respective tissue elements post injury. Data were analyzed using Students t test and analysis of variance with P ≤ 0.05 considered statistically significant. Results Addition of 1.2% BaCl2 to PSS depolarized myofibers from − 79 ± 3 mV to − 17 ± 7 mV with a corresponding rise in [Ca2+]i; isometric force transiently increased from 7.4 ± 0.1 g to 11.1 ± 0.4 g. Following 1 h of BaCl2 exposure, 92 ± 3% of myonuclei stained with PI (vs. 8 ± 3% in controls) with enhanced cleavage of αII-spectrin. Eliminating Ca2+ from PSS prevented the rise in [Ca2+]i and ameliorated myonuclear staining with PI during BaCl2 exposure. Motor axons and capillary networks appeared fragmented within 24 h following injection of 1.2% BaCl2 and morphological integrity deteriorated through 72 h. Conclusions BaCl2 injures myofibers through depolarization of the sarcolemma, causing Ca2+ overload with transient contraction, leading to proteolysis and membrane rupture. Motor innervation and capillarity appear disrupted concomitant with myofiber damage, further compromising muscle integrity.


Background
Acute injury to skeletal muscle initiates a coordinated process of tissue degeneration and regeneration that encompasses inflammation; digestion of damaged components; activation, proliferation, and differentiation of resident myogenic stem cells (satellite cells); and maturation of nascent myofibers [1,2]. While different injury models (freeze injury, cardiotoxin, Marcaine™, and BaCl 2 ) induce variable degrees of tissue damage and inflammation [3][4][5], the advantages of chemical injury with BaCl 2 include both ease of use and its ability to reproducibly damage myofibers while preserving their associated satellite cells [3,[6][7][8][9] (Additional file 1). However, the mechanism by which BaCl 2 exposure leads to the death of skeletal muscle myofibers has not been identified. The divalent cation Ba 2+ blocks inward rectifying potassium channels (K IR ) at concentrations of 10-100 μM [10] and serves as a broad spectrum K + channel inhibitor at concentrations ≥ 1 mM [11,12]. Thus, injection of 1.2% BaCl 2 (~57 mM), as used to induce muscle damage [3,[6][7][8][9], would be predicted to depolarize myofibers. In turn, depolarization can activate L-type voltage-gated calcium channels (Ca V 1.1) in the sarcolemma, leading to increases in intracellular Ca 2+ concentration ([Ca 2+ ] i ) via release from the sarcoplasmic reticulum and influx from the extracellular fluid [13,14]. Sufficient elevation of [Ca 2+ ] i initiates proteolysis, leading to degradation of contractile proteins and cell membranes [15,16]. We therefore tested the hypothesis that BaCl 2 injures skeletal muscle through myofiber depolarization, with elevated [Ca 2+ ] i leading to proteolysis and rupture of the sarcolemma.
Motor axons and microvessels are intimately associated with myofibers. At the neuromuscular junction (NMJ), myelinated axons projecting from α motor neurons in the spinal cord terminate and are covered by perisynaptic Schwann cells which overlay postsynaptic clusters of nicotinic acetylcholine receptors [17]. Arterioles control the perfusion of capillary networks that collectively span the entire length of myofibers to provide oxygen and nutrients essential to supporting contractile activity [18]. While BaCl 2 can damage the capillary supply [3], it is unknown whether concomitant injury occurs to the motor nerves that control myofiber contraction. Therefore, also we tested whether local injection of BaCl 2 disrupts the integrity of NMJs and capillaries concomitant with injuring myofibers.

Aim, design, and setting
The aim of this study was to determine how BaCl 2 injures skeletal muscle myofibers and whether motor innervation and microvascular supply are concomitantly disrupted by exposure to BaCl 2 . We studied the acute effects of BaCl 2 exposure on membrane potential (V m ), [Ca 2+ ] i , membrane integrity, αII-spectrin degradation, and force production in extensor digitorum longus (EDL) muscles. Neuromuscular synapses were studied in the tibialis anterior (TA) muscle and the microcirculation was studied in the gluteus maximus (GM) muscle at 0 (control) and 1-3 days post injury (dpi) following local injection of BaCl 2 .
To induce muscle injury in vivo, mice were anesthetized with ketamine and xylazine (100 mg/kg and 10 mg/ kg, respectively; intraperitoneal injection), the skin was shaved over the muscle of interest, and then 1.2% BaCl 2 was injected unilaterally into the TA (50 μL [9]) or under the GM (75 μL [8]) as described. Mice were kept warm during recovery and then returned to their cage. On the day of an experiment, mice were anesthetized (as above). Following tissue harvest, mice were killed by exsanguination.

Membrane potential
The EDL muscle was used for these experiments because it can be isolated and secured in vitro by its tendons to approximate in vivo muscle length without damaging myofibers. An EDL muscle was removed from the anesthetized mouse, pinned onto transparent rubber (Sylgard 124; Midland, MI, USA), placed in a tissue chamber (RC-37 N; Warner Instruments; Hamden, CT, USA), transferred to the stage of a Nikon 600FN microscope (Tokyo, Japan), and irrigated (3 mL min − 1 ) with standard physiological salt solution (PSS; pH 7.4) of the following composition: 140 mM NaCl (Fisher Scientific; Pittsburgh, PA, USA), 5 mM KCl (Fisher), 1 mM MgCl 2 (Sigma), 10 mM HEPES (Sigma), 10 mM glucose (Fisher), and 2 mM CaCl 2 (Fisher) while maintained at 37°C.
The membrane potential (V m ) of myofibers was recorded with an amplifier (AxoClamp 2B, Molecular Devices; Sunnyvale, CA, USA) using sharp microelectrodes pulled (P-97, Sutter Instruments; Novato, CA, USA) from glass capillary tubes (GC100F-10, Warner, Hamden, CT, USA) filled with 2 M KCl (~150 MΩ) with a Ag/AgCl pellet serving as a reference electrode [20]. The amplifier was connected to a data acquisition system (Digidata 1322A, Molecular Devices) and an audible baseline monitor (ABM-3, World Precision Instruments; Sarasota, FL, USA). Successful impalements were indicated by sharp negative deflection of V m , stable V m for > 1 min, and prompt return to~0 mV upon withdrawal of the electrode. Data were acquired at 1 kHz on a personal computer using AxoScope 10.1 software (Molecular Devices). Once a single myofiber was impaled, V m was recorded for at least 5 min to establish a stable baseline. PSS containing 1.2% BaCl 2 then irrigated the muscle at 37°C. Additional experiments were performed using isotonic substitution of BaCl 2 for NaCl (final [NaCl] = 54 mM vs. 140 mM in standard PSS) to test whether differences in osmolality affected responses during exposure to 1.2% BaCl 2 . Each of these experiments represents one myofiber in one EDL; each muscle was obtained from a separate mouse.

Membrane damage
As an index of myofiber membrane damage, EDL muscles were treated for 1 h with standard PSS, 1.2% BaCl 2 dissolved in standard PSS, or 1.2% BaCl 2 dissolved in Ca 2+free PSS then stained for 20 min with membranepermeant Hoechst 33342 (1 μM, Cat. # H1399, Fisher) and membrane-impermeant propidium iodide (PI; 2 μM, Cat. # P4170, Sigma) in PSS. These dyes stain the nuclei of all cells and nuclei of cells with disrupted membranes, respectively [21]. Muscles were then washed for 30 min in standard PSS and image stacks were acquired with a water immersion objective (× 40; NA = 0.8) coupled to a DS-Qi2 camera with Elements software (version 4.51) on an E800 microscope (all from Nikon). Stained nuclei were counted within a defined region of interest (ROI; 300 × 400 μm) of image stacks using Image J (NIH) to quantify the percentage (%) of total nuclei stained with PI.
Western blot for αll-spectrin degradation EDL muscles were secured to approximate in situ length and incubated in either standard PSS or 1.2% BaCl 2 in PSS for 1 h at 37°C then frozen in liquid nitrogen. Following homogenization, protein concentration of the supernatant was quantified with the Bradford method (Cat. # 5000006; Sigma). Protein concentration of each sample was normalized in 4x Laemmli sample buffer (Cat. # 1610747, Bio-Rad; Hercules, CA, USA) containing 5% dithiothreitol. Samples were loaded on 4-20% gradient Mini-Protein TGX gels (Bio-Rad) for electrophoresis and transferred to LF-PVDF membranes (Millipore; Burlington, MA). Following 2 h blocking in 5% milk, membranes were incubated overnight at 4°C and again for 3 h at 25°C in primary antibody raised against αII-spectrin (1:250, Cat. # sc48382, Santa Cruz; Dallas, TX, USA). A secondary antibody (Alexa Fluor 800 IgG, 1:5000; Cat. # 926-32,212, Li-Cor Biosciences; Lincoln, NE, USA) was used to quantify protein differences with an Li Cor Odyssey Fc imaging system. Western blots were normalized to total protein according to the recommendations for fluorescent Western blotting [22] using Revert total protein stain (Cat. # 926-11010, Li-Cor). The 40 kDa bands correlate with the total protein in each lane and are shown to represent equal protein loading [23,24].

Muscle force
The EDL was prepared for in situ measurements as described [25]. Briefly, in an anesthetized mouse, a 2-0 suture was placed around the left patellar tendon. The distal tendon of the EDL was isolated, secured in 2-0 suture, and then severed from its insertion. The mouse was placed prone on a plexiglass board and the patellar tendon was secured to a vertical metal peg immobilized in the board. The distal EDL tendon was tied to a load beam (LCL-113G; Omega, Stamford, CT, USA) coupled to a Transbridge amplifier (TBM-4; World Precision Instruments, Sarasota, FL, USA). The load beam was attached to a micrometer for adjusting optimal length (L o ) as determined during twitch contractions at 1 Hz [8]. A strip of KimWipe® was wrapped around the EDL and 1.2% BaCl 2 irrigated the EDL (3 mL min − 1 ) while resting force was evaluated for 1 h with Power Lab acquisition software (ADInstruments, Colorado Springs, CO, USA) on a personal computer.

Neuromuscular junction histology
In a mouse strain with genetically labeled Schwann cells (S100B-GFP/Kosmos [26]), the TA muscle of one hindlimb was injured with BaCl 2 injection and the contralateral limb was left intact. Mice were studied at 0 (control), 1, 2, and 3 dpi. At each time point, the hindlimb was excised, the TA was removed, and myofibers were gently teased apart with fine forceps in ice-cold phosphate-buffered saline (PBS, pH 7.4) to facilitate antibody penetration. Samples were fixed for 15 min in 4% paraformaldehyde, washed in PBS 3 times for 5 min, and stained for neurofilament-heavy (primary antibody: chicken anti-mouse, 1:400; Cat. # CPCA-NF-H Encor Biotechnology Inc.; Gainesville, FL, USA; secondary antibody: goat anti-chicken, 647 IgY, 1:1000, Cat. # A-21449, Fisher); each antibody was incubated overnight at 4°C followed by washing in PBS 6 times for 30 min. Nicotinic receptors were then stained with α-bungarotoxin conjugated to tetramethylrhodamine (1:500, Cat. # 00014, Biotrend; Koln, Germany) for 2 h at room temperature and washed in PBS prior to imaging. Images were acquired with a × 25 water immersion objective (NA = 0.95) at × 1.75 digital zoom on an inverted laser scanning confocal microscope (TCS SP8, Leica Microsystems Buffalo Grove, IL, USA) using Leica LAX software. Image stacks (thickness,~150 μm) were used to resolve NMJ morphology.

Microvessel histology
The GM was used for histological analysis of skeletal muscle microvasculature based on it being a thin (100-200 μm), planar muscle which facilitates imaging of microvessels throughout the tissue [8]. A GM was dissected away from its origin along the lumbar fascia, sacrum, and iliac crest, reflected away from the body, and spread onto a transparent rubber pedestal. Superficial connective tissue was removed using microdissection and the muscle was severed from its insertion. To image capillary networks, the unfixed GM was immersed in PBS, a small glass block was placed on top to gently flatten the muscle, and image stacks were acquired as described for NMJs. In Cdh5-mTmG mice, all endothelial cells are labeled with membrane-localized GFP following tamoxifen-induced Cre recombination.

Data analysis
Data were analyzed using Student's t test and one-way Analysis of Variance with Bonferroni's multiple comparison test post hoc when appropriate (Prism 5, GraphPad Software, La Jolla, CA, USA). Summary data are presented as means ± SEM; n refers to the number of preparations (each from a different mouse) in a given experimental group. P ≤ 0.05 was considered statistically significant.
Irrigating the EDL in situ with 1.2% BaCl 2 in standard PSS increased resting force from 7.4 ± 0.1 to 11.1 ± 0.4 g over~30 min, which then returned to baseline during the 60 min exposure ( Fig. 2b; P = 0.001). Whereas a rise in [Ca 2+ ] i activates the contractile proteins [32], sustained elevation of [Ca 2+ ] i stimulates mitochondrial production of reactive oxygen species (ROS), which can impair cross-bridge function [33]. Ca 2+ -activated proteolysis disrupts the integrity of contractile proteins [15], which we surmise may have occurred in the present experiments.

BaCl 2 induces injury of motor axons and microvessels
In contrast to the integrity of pre-and postsynaptic elements characteristic of healthy NMJs, neurofilamentheavy staining at 1 dpi appeared fragmented, suggesting axonal disruption. Clusters of acetylcholine receptors also became dispersed along the laminar surface and Schwann cells began to migrate away from the NMJ, which progressed through 2 dpi (Fig. 5a). By 3 dpi, Schwann cells appear to associate with axonal fragments and AChR clusters. These data demonstrate that motor axons in the vicinity of BaCl 2 injection undergo degeneration within 24 h that extends over 3 days, consistent with the time course of Schwann cell migration following axotomy [39].
Uninjured muscle exhibits an orderly network of capillaries (Fig. 5b). Following BaCl 2 injury, capillaries were fragmented at 1 dpi. By 3 dpi, anastomoses (interconnecting loops) began to appear between capillary sprouts. While these observations add new insight to the extent of tissue injury induced by BaCl 2 , our findings are consistent with structural damage of microvessels induced by BaCl 2 at 2 dpi [3] and our recent report that capillary perfusion was disrupted at 1 dpi [8].

Discussion
Skeletal muscle comprises~40% of body mass and has the remarkable ability to regenerate following injury due to resident satellite cells. Skeletal muscle injuries occur in multiple ways including disease, physical trauma, temperature extremes, eccentric contractions, and exposure to myotoxic agents [3][4][5]. Whereas myofibers follow a similar pattern of regeneration irrespective of the mechanism of injury [1,40], the kinetics and involvement of satellite cells can vary with the nature of insult b Summary data for V m are at resting baseline, at peak depolarization during 1.2% BaCl 2 added to standard PSS and to PSS in which BaCl 2 replaced NaCl for osmotic (Osm) control. c Summary data for time to peak depolarization during 1.2% BaCl 2 added to standard PSS, and to PSS in which BaCl 2 replaced NaCl for Osm control. Values are means ± SEM (n = 3-6 myofibers, each from one EDL muscle per mouse). # P ≤ 0.05 vs. baseline [3]. For example, freeze injury results in a dead zone of tissue that viable cells must penetrate, whereas local exposure to BaCl 2 induces coordinated necrosis of myofibers with infiltration of inflammatory cells followed by sequential regeneration of myofibers [1,3].
Unlike freeze damage, BaCl 2 -induced injury preserves satellite cells, which allows detailed examination of their gene expression, cell signaling, and regeneration kinetics in vivo. Remarkably, how BaCl 2 kills myofibers has remained undefined. In accord with the ability of Ba 2+ to block K + channels [11,12], we reasoned that it would depolarize myofibers, as the sarcolemma contains K V , K IR , K Ca , and K ATP channels [30]. The progression of depolarization we observed in EDL may reflect reliance on Cl − conductance for resting V m in skeletal muscle, which can buffer the abrupt effect of changing the conductance of other ions [41]. Following BaCl 2 -induced depolarization, the present data show that increasing [Ca 2+ ] i leads to proteolysis, membrane disruption, and myofiber death. Moreover, preventing the rise of [Ca 2+ ] i by removing extracellular Ca 2+ preserves membrane integrity as demonstrated by the paucity of myonuclei stained with PI under this condition (Fig. 4c). Myotoxicity through Ca 2+ -mediated proteolysis and membrane disruption subsequent to Ba 2+ exposure is consistent with the action of biological agents known to disrupt myofibers such as bee, wasp, and snake venoms [42][43][44]. BaCl 2 has been used to study the pathophysiology of hypokalemia, a clinical condition which depolarizes muscle fibers through reduced K + efflux [10,45]. In hypokalemia, the SR is integral to myofiber disruption [10,46,47], with Ca 2+ release from internal stores being a primary source of the elevated [Ca 2+ ] i that contributes to muscle injury. While the relative contribution of Ca 2+ release from internal stores vs. influx through L-type channels during BaCl 2 injury remains to be determined, the SR is the principal source of elevating [Ca 2+ ] i during muscle contractions [48]. Consistent with this effect, we observed transient contraction of the EDL upon exposure to BaCl 2 that peaked with the rise in [Ca 2+ ] i over 20-30 min (Fig. 2). The recovery to resting (passive) tension during the ensuing 30 min may reflect disruption of the contractile machinery. This interpretation is consistent with the degradation of αII-spectrin we observed within 60 min of BaCl 2 exposure (Fig. 3). Once in the cytoplasm, Ba 2+ can enter mitochondria [49] and generate superoxide by increasing electron flow from Ca 2+ -sensitive citric acid cycle dehydrogenases and thereby dissipate mitochondrial membrane potential [50]. The ensuing disruption of mitochondria releases cytochrome C into the cytosol to initiate intrinsic apoptosis, culminating in the activation of caspase 3 and cell death [36].

Nerve and microvessel injury
Motor nerves and microvessels control and supply myofibers of intact skeletal muscle by initiating contraction and delivering nutrients in response to metabolic demand [51]. Given their intimate physical proximity and shared signaling events, we hypothesized that muscle injury induced by BaCl 2 would disrupt motor axons and capillaries. Similar to the time course of myofiber disruption [3], the present data illustrate that motor nerves and microvessels appear fragmented within 24 h following local injection of BaCl 2 (Fig. 5).
It is unclear whether nerves and capillaries undergo damage directly from BaCl 2 or indirectly as a secondary effect of myofiber disruption. Mechanical changes within the injured myofiber can lead to degeneration of the NMJ. For example, with local injury, myofilaments contract on both sides of the injured site, leaving an empty tube with partially retracted nerve terminals juxtaposed  Representative Western blots (top) and mean densitometric data (bottom) for αII-spectrin from EDL muscles treated with standard PSS (Control) or 1.2% BaCl 2 in standard PSS for 1 h. The αII-spectrin band at 240 kDa and its cleavage product at 150 kDa were both normalized to total protein reflected by the 40 kDa band, which was not different between samples. Summary data are means ± SEM (n = 6 muscles). # P ≤ 0.05 vs. control to the site [52]. While mechanisms of axon retraction remain to be defined, the change in cell shape suggests that it is a consequence of cytoskeletal remodeling in response to a retraction program or loss of the ability to maintain the cytoskeleton [53]. Because it is a cytoskeletal protein integral to the structure of cell membranes, degradation of αII-spectrin is disruptive to the sarcolemma and contributes to fragmentation of motor nerve synapses with dissolution of AChR clusters (Fig. 5).
Disruption of capillaries occurs in multiple models of muscle injury [3]. The present data are the first to illustrate that these events coincide with the loss of neuromuscular integrity. Thus, key elements of myofiber control and supply are similarly affected, with loss of structural integrity occurring during the initial 24 h (1 dpi) and initial stages of recovery apparent at 3 dpi. In addition to myofibers, vessels and nerves may undergo calpain-dependent degradation [54,55]. Thus, while skeletal muscle consists primarily of myofibers, the increase in calpain-specific αII-spectrin degradation measured in our homogenates (Fig. 3) may be derived from multiple cell types. As we have observed directly with intravital microscopy in the GM, the inflammatory response to BaCl 2 begins within 1-2 h of exposure (Fernando and Segal, unpublished observations from [8]). Infiltration of the tissue with neutrophils, monocytes, and pro-inflammatory macrophages ensues over the next 2-3 days, thereby disrupting all tissue components indiscriminately [3,56] through activation of additional proteolytic pathways and oxidative modification of proteins to accelerate proteolysis [36].

Conclusion
Skeletal muscle injury induced by BaCl 2 is widely used as a method for studying myofiber damage and regeneration [3,[6][7][8][9]. Because the mechanism of BaCl 2 -induced injury was unknown, the goal of the present study was to define the nature of myofiber damage and ascertain whether associated tissue elements were similarly affected. Using complementary ex vivo preparations of skeletal muscle, we demonstrate that acute exposure to BaCl 2 causes myofiber damage via Ca 2+ -dependent proteolysis secondary to membrane depolarization. Further, motor axons and microvessels appear to undergo damage with a similar time course to the disruption of myofibers. These data provide a foundation for investigating how major tissue components responsible for skeletal muscle structure and function (i.e., myofibers, motor nerves, and microvessels) respond to and interact during muscle injury and regeneration. Motor innervation and capillaries are disrupted by local BaCl 2 injection. a Neuromuscular junctions in TA muscle. Schwann cells (green) are closely associated with axonal neurofilament-heavy (cyan; indicates motor axons) and overlay postsynaptic nicotinic receptors (red) at 0 dpi (uninjured control). Following injection of 1.2% BaCl 2 , NMJ components are dissociating at 1 dpi and fragmented at 2 and 3 dpi. b Capillaries in GM (green endothelial cells) are densely organized and align along myofibers in uninjured muscle (0 dpi). Following injection of 1.2% BaCl 2 , disrupted and fragmented capillaries are observed at 1-2 dpi (arrowheads) while evidence of capillary neoformation is apparent at 3 dpi (arrowhead). Scale bars, 50 μm. Color coding in 3 dpi panels applies to earlier timepoints for innervation and capillarity. NF-H, neurofilament heavy; AChR, nicotinic actetylcholine receptors; S100B, Schwann cells expressing GFP; Cdh5, endothelial cells expressing GFP