Functional β-Adrenoceptors Are Important for Early Muscle Regeneration in Mice through Effects on Myoblast Proliferation and Differentiation

Muscles can be injured in different ways and the trauma and subsequent loss of function and physical capacity can impact significantly on the lives of patients through physical impairments and compromised quality of life. The relative success of muscle repair after injury will largely determine the extent of functional recovery. Unfortunately, regenerative processes are often slow and incomplete, and so developing novel strategies to enhance muscle regeneration is important. While the capacity to enhance muscle repair by stimulating β2-adrenoceptors (β-ARs) using β2-AR agonists (β2-agonists) has been demonstrated previously, the exact role β-ARs play in regulating the regenerative process remains unclear. To investigate β-AR-mediated signaling in muscle regeneration after myotoxic damage, we examined the regenerative capacity of tibialis anterior and extensor digitorum longus muscles from mice lacking either β1-AR (β1-KO) and/or β2-ARs (β2-KO), testing the hypothesis that muscles from mice lacking the β2-AR would exhibit impaired functional regeneration after damage compared with muscles from β1-KO or β1/β2-AR null (β1/β2-KO) KO mice. At 7 days post-injury, regenerating muscles from β1/β2-KO mice produced less force than those of controls but muscles from β1-KO or β2-KO mice did not exhibit any delay in functional restoration. Compared with controls, β1/β2-KO mice exhibited an enhanced inflammatory response to injury, which delayed early muscle regeneration, but an enhanced myoblast proliferation later during regeneration ensured a similar functional recovery (to controls) by 14 days post-injury. This apparent redundancy in the β-AR signaling pathway was unexpected and may have important implications for manipulating β-AR signaling to improve the rate, extent and efficacy of muscle regeneration to enhance functional recovery after injury.


Introduction
Skeletal muscle is susceptible to damage associated with trauma, metabolic deficits, heritable and acquired diseases, and mechanical strains generated during contractions [1][2][3][4]. Although skeletal muscle has an impressive ability to regenerate after injury, the repair process is often slow and incomplete, compromised by factors such as age and pre-existing health conditions [5]. To develop treatment strategies that enhance muscle regeneration after injury and thereby improve quality of life for patients, a more comprehensive understanding of the events taking place during the regeneration of skeletal muscle is required [6].
Many of the most common and important growth factors and hormones can influence the attributes of mature and regenerating skeletal muscles. For instance, we and others have shown that stimulating b-adrenoceptors (b-ARs) with b-AR agonists (bagonists) can enhance skeletal muscle repair after injury [6][7][8][9]. Recent studies have also shown that G ai2 , a component of the b-AR signalling pathway, plays an important role in regeneration after injury and general maintenance of muscle mass [10][11][12]. However, while the capacity to enhance muscle repair via exogenous stimulation of b-ARs is now recognised, it remains unclear as to the role b-ARs play in regulating the regenerative process. To elucidate the significance of b-AR-mediated signalling in regeneration, we examined the regenerative capacity of limb muscles from mice lacking either b 1 -AR and/or the b 2 -AR. On the basis that exogenous administration of b 2 -agonists can promote regeneration, it was hypothesised that muscles from mice lacking the b 2 -AR (b 2 -KO) would exhibit impaired functional regeneration after damage compared with muscles from b 1 -KO or b 1 /b 2 -KO mice.

Animals
All procedures were approved by the Animal Ethics Committee of The University of Melbourne and conformed to the Australian code of practice for the care and use of animals for scientific purposes as stipulated by the National Health and Medical Research Council of Australia. Mice lacking both the b 1 -and b 2 -AR (b 1 /b 2 -KO) were purchased from The Jackson Laboratory (Adrb1 tm1Bkk Adrb2 tm1Bkk/j , stock #003810) and maintained by mating double homozygote KO mice. Control mice were from a C57BL/6 background, as employed previously for comparison with b 1 /b 2 -KO [13,14] and obtained from the Animal Resources Centre (Canning Vale, WA, Australia). Mice lacking either the b 1 -AR or the b 2 -AR were purchased from The Jackson Laboratory and back crossed with C57BL/6 mice to create a heterozygous F1 generation. The heterozygous offspring of the respective strains were subsequently inbred to produce the respective b 1 -KO and b 2 -KO knockout animals and littermate controls (b 1 -WT and b 2 -WT, respectively). Mice were housed in the Biological Research Facility at The University of Melbourne under a 12 h light-dark cycle, with drinking water and standard chow provided ad libitum. Male mice aged 8-9 weeks were used in this study.

Myotoxic injury
Injury was induced in the tibialis anterior (TA) or extensor digitorum (EDL) muscles of mice as described previously [15,16]. For TA injuries, mice were anesthetized using a mixture of ketamine (76 mg/kg, i.p.) and xylazine (10 mg/kg, i.p.) and a small portion of the TA muscle of the right hindlimb was surgically exposed by a single incision through the skin. The muscle was filled to its maximal holding capacity (,40 ml) via a single intramuscular injection with Notexin (1 mg/ml, Latoxan) using a 29-gauge needle. The wound was closed with Michel clips (Aesculap, Tuttlingen, Germany) and the mice were allowed to recover from the myotoxic injury for 7, 10 or 14 days before assessments of muscle structure and function were performed. Uninjured mice were used as controls for all experiments using the TA muscle. For EDL injuries, mice were anesthetized as described and the EDL muscle of the right hindlimb was surgically exposed by a single incision through the skin and overlying muscles. With the aid of a dissecting microscope, the EDL was filled to its maximal holding capacity (15-20 ml) via intramuscular injection at several sites with Notexin (1 mg/ml, Latoxan) using a 30-gauge needle. The wound was closed with Michel clips (Aesculap, Tuttlingen, Germany) and the mice were allowed to recover from the myotoxic injury for 2, 5, 7, 14 or 21 days. The corresponding muscle from the left (contralateral) limb served as the non-injured control muscle.

Assessment of muscle function
Mice were anesthetized with sodium pentobarbital (60 mg/kg, i.p.) and muscle function was assessed either in vitro (for EDL) or in situ (for TA) as described previously [17,18]. For assessment of TA muscle force producing capacity in situ, the muscles of anesthetised mice were stimulated by supramaximal 0.2 ms square wave pulses of 350 ms duration, delivered via two wire electrodes placed adjacent to the deep peroneal branch of the sciatic nerve. Force generated was measured via attachment of a force transducer to the distal tendon of the muscle. For assessments of EDL muscle function in vitro the muscles from each limb were surgically excised from anesthetized mice and transferred to a custom-built organ bath filled with Krebs-Ringer solution (in mM: NaCl, 1.37; NaHCO 3 , 24; D-glucose, 11; KCl, 5; CaCl 2 , 2; NaH 2 PO 4 , 1; MgSO 4 , 0.487, pH 7.4) supplemented with d-tubocurarine chloride (0.293 mM), bubbled with Carbogen (5% CO 2 in O 2 ) and maintained at 25uC, where the proximal tendon was attached to a micro-manipulator (to facilitate adjustment of muscle length to optimal), and the distal tendon was attached to a force transducer. The EDL was stimulated by supramaximal (40 V) 0.2 ms square wave pulses of 350 ms duration delivered via platinum plate electrodes that flanked the full length of each muscle.
For both in situ and in vitro assessments, optimal muscle length for contraction (L o ) was defined as the muscle length at which maximal isometric twitch force (P t ) was attained and maximal isometric tetanic force (P o ) was determined from the plateau of the frequency-force relationship. P o was normalized to the crosssectional area of the muscle (CSA; calculated using L o and muscle mass) in order to determine maximum specific isometric tetanic force (sP o ), a measurement that allows for relative comparisons of force production between muscles of different sizes.
Immediately following the functional assessments, muscles were carefully trimmed of any adherent non-muscle tissue and tendons, and weighed on an analytical balance. Muscles were then mounted in Tissue-Tek OCT embedding medium, frozen rapidly in thawing isopentane, and stored at -80uC for later histological and biochemical analyses. The mice were killed by surgical excision of the heart while still anesthetized deeply.

Muscle morphology
Serial transverse cryosections (5 mm) were cut from the midbelly of each muscle and placed onto glass slides (Superfrost Plus, Menzel-Glä ser, Kensington, VIC, Australia). General muscle histology was determined by staining sections with hematoxylin and eosin (H & E) to visualize muscle fibers, and digital images of stained sections were obtained using a microscope equipped with digital camera (Carl Zeiss, Wrek, Göttingen, Germany) supported by associated imaging software (Axiovision V4.7.1.0).

Quantitative RT-PCR
Muscle samples were homogenized individually and mRNA extracted using an RNEasy fibrous tissue RNA extraction kit (Qiagen), according to manufacturer's instructions. The concentration and quality of RNA in each sample was determined using a Nanodrop 2000 (Thermo Scientific) and the extracted mRNA was stored at 280uC. mRNA was transcribed into cDNA using the Superscript VILO cDNA synthesis kit (Invitrogen) according to manufacturer's instructions, and stored at 220uC until use. Quantitative RT-PCR was performed using an iCycler Thermal Cycler (Bio-Rad) with SYBR Green supermix (Quantace). Primers were designed using the Perfect Primer online program (Invitrogen) and are listed in Table 1. Due to the severity of the myotoxic damage caused by Notexin at the earlier time points [15], we chose not to use the DCt method of analysis as we could not be certain that any reference gene used would remain unchanged between treatment groups. We instead opted to measure the cDNA concentration of each sample using the Quant-iT OliGreen ssDNA Assay Kit (Molecular Probes) and to normalize our data to cDNA content as described previously [19,20]. anesthetized and killed by cardiac excision and the quadriceps, TA and gastrocnemius muscles were excised, cleaned of all connective tissue, and rinsed in ice-cold culture media (Ham's Nutrient Mix F-12). The muscles were placed in a digest solution consisting of culture media supplemented with 1% Type II collagenase and 0.5% Dispase (Invitrogen), minced with a clean pair of scissors, and incubated in a shaking incubator at 37uC for 90 min. The suspension was filtered through a 100 mm cell filter (BD Biosciences), pelleted (15006g, 5 min) and resuspended in complete growth media (Ham's Nutrient Mix F-12 supplemented with 20% FBS, 1% L-glutamine, 1% antibiotic-antimycotic and 5 ng/ml rhFGF). The cell suspension was plated into uncoated cell culture dishes overnight to allow fibroblasts to adhere, after which the non-adherent cells were aspirated and plated into a fresh uncoated dish for a further 24 hrs to remove as many fibroblasts as possible. Following the second overnight incubation, non-adherent cells were aspirated and seeded into a cell culture dish pre-coated with extracellular matrix (ECM; Sigma). Cells were expanded in culture for approximately 4 weeks, after which myoblast proliferation and differentiation were assessed.
For proliferation assays, isolated primary myoblasts were seeded into ECM-coated 96 well plates at a density of 1610 4 cells/well and incubated in complete growth media for 72 hrs. Subsequently, the cells were fixed (3.7% formaldehyde, 10 min, 25uC), rinsed twice with PBS, and incubated with DAPI (0.235 mM diluted in PBS, 15 min, 25uC). The number of cell nuclei was quantified using an inverted fluorescence microscope and camera (Axiovert 40, Carl Zeiss) and associated imaging software (Axiovision V4.7.1.0, Carl Zeiss).
For differentiation assays, the isolated primary myoblasts were seeded into ECM-coated 12-well plates at a density of 5610 5 cells/ well. After incubating for 2 hr to permit cell attachment, growth media was replaced with differentiation media (DMEM supplemented with 2% horse serum and 1% L-glutamine). Cells were cultured in differentiation-inducing conditions for 3 days, with differentiation media replaced daily. To examine attributes of differentiation, cells were imaged with an inverted microscope and camera (Axiovision 40, Carl Zeiss) controlled by associated software (Axiovision V4.7.1.0). Differentiation was measured as total area (mm 2 ) in the field of view occupied by multinucleated myotubes.

Statistical analyses
All values are reported as mean 6SEM. Groups were compared using an unpaired Student's t-test, or a one-way or two-way ANOVA and Bonferroni's post hoc multiple comparison procedure where appropriate. Myofiber cross-sectional area (CSA) is not normally distributed, and so medians were compared using a nonparametric Mann-Whitney test by ranks. All statistical analyses were performed using Prism version 3 software (GraphPad Software, Inc. La Jolla, CA). In all cases significance was defined as P,0.05.

Results
Deletion of specific b-ARs has differential effects on muscle mass and force producing capacity We first compared the morphology and functional characteristics of TA muscles from the different b-AR KO strains of mice. We found no significant differences in body mass between any of the b-KO mice and their respective controls (Fig. 1A). When we compared the muscle mass between the strains, neither b 1 -KO mice nor b 2 -KO mice were different from their respective controls, but TA muscles of b 1 /b 2 -KO mice were significantly smaller (Fig. 1B). When maximal force was expressed as absolute force (P o ), b 1 -KO mice did not exhibit differences in force production compared with controls, but both b 2 -KO mice and b 1 / b 2 -KO mice produced less force than their controls (Fig. 1C). When maximal force production was normalized to muscle size (sP o ), b 1 -KO mice still exhibited no differences in force production and b 2 -KO mice still exhibited deficits in force production, but b 1 /b 2 -KO mice demonstrated equivalent functional capacity compared with control ( Fig. 1D). Examining muscle fiber crosssectional area (CSA) for the various b-KO mouse strains revealed that, as for TA muscle mass, neither the muscles of b 1 -KO mice  nor b 2 -KO mice were different from the muscles of their respective littermate controls, but that muscle fiber CSA in b 1 / b 2 -KO mice was significantly smaller than muscle fibres from wild type mice (Fig. 1E). We also analyzed a number of twitch characteristics of TA muscles from the b-KO mice ( Table 2) and found that the muscles of b 1 -KO mice did not differ from control mice, but the muscles of both b 2 -KO mice and b 1 /b 2 -KO mice elicited reduced peak twitch force (P t ) and a reduced rate of contraction (dP t /dt) compared with the muscles of controls. An analysis of the frequency-force relationship found no significant differences between b 1 -KO mice and control, but significant differences between b 2 -KO mice and control, and b 1 /b 2 -KO mice and control ( Figure S2).

Deletion of b 2 -ARs impairs the functional performance of regenerating muscles
We next examined the recovery of force production in the TA muscles of the various b-KO mice at 7, 10 and 14 days after myotoxic damage. When force production was expressed as absolute force (P o ), we found that b 1 -KO mice displayed no differences in maximal force production during regeneration, whereas both b 2 -KO mice and b 1 /b 2 -KO mice produced significantly less force compared with controls ( Figure S1). When force was normalized to muscle size (sP o ) maximal force production was unchanged in b 1 -KO mice from controls ( Fig. 2A). Maximal force in b 2 -KO mice was significantly lower than controls throughout regeneration (Fig. 2C), but force production by b 1 /b 2 -KO mice was no longer significantly different from control over the 14 days of regeneration (Fig. 2E). In order to take into account the lower sP o of uninjured muscles from b 2 -KO mice, we then expressed force production during regeneration as a percentage of that produced by uninjured muscles (%UI). When maximal force was expressed this way, we found that none of the b-KO strains exhibited any deficits in the restoration of force producing capacity compared with controls ( Fig. 2B, 2D and 2F). When the data at 7 days post-injury were analyzed separately in order to assess the earliest time point in isolation, b 1 /b 2 -KO mice had impaired force production compared with controls whether expressed as P o , sP o or %UI.
In order to more comprehensively assess the role of b-ARs in skeletal muscle we also compared morphological characteristics of EDL muscles between b 1 /b 2 -KO and C57BL/6 control mice. Our findings for the EDL muscles closely mirrored those for the TA muscle; viz., muscle fiber CSA was not significantly different between b 1 /b 2 -KO mice and C57BL/6 controls (Fig. 3A) and uninjured muscles from b 1 /b 2 -KO mice were significantly smaller than controls (Fig. 3B) and produced lower maximal absolute forces ( Fig. 3c and Table 3). Similarly, when maximal force was normalized to muscle cross-sectional area (sP o ) there were no significant differences between b 1 /b 2 -KO mice and controls ( Fig. 3D and Table 3). When twitch characteristics of uninjured EDL muscles were compared between the two strains, we found that as for the TA muscles, both twitch force (P t ) and rate of contraction (dP t /dt) were significantly lower in the b 1 /b 2 -KO mice (Table 3). Following myotoxic damage, EDL muscles from b 1 /b 2 -KO mice exhibited impaired restoration of force production over 21 days, with a particularly significant deficit at 7 days post-injury ( Fig. 3C and 3D). In addition to the delayed restoration of contractile force in recovery EDL muscles, qualitative differences in muscle architecture were observed at both 7 and 14 days post-injury (Fig. 3E).
Expression of inflammatory markers and myogenic regulatory factors during early regeneration is potentiated in muscles lacking b 1 -and b 2 -ARs As we had detected impaired force production in b 1 /b 2 -KO mice during the early stages of regeneration (at 7 days post-injury), we examined the expression of both inflammatory markers and myogenic regulatory factors in TA muscles from b 1 /b 2 -KO mice and C57BL/6 control mice immediately after myotoxic injury and during early regeneration. Quantitative RT-PCR for the cytokines interleukin-6 (IL-6) and tumour necrosis factor-a (TNF-a) revealed an increase in cytokine expression in C57BL/6 mice after myotoxic damage, which peaked at 2 days post-injury ( Fig. 4C  and 4D). Injury-induced expression of TNF-a was significantly higher in b 1 /b 2 -KO mice than in C57BL/6 controls ( Fig. 4D; 2way ANOVA, main strain effect), and expression of IL-6 showed a non-significant trend toward higher expression in b 1 /b 2 -KO mice (Fig. 4C). Expression of the macrophage markers CD68 and F4/ 80 revealed an increase in macrophage infiltration in injured muscles from both b 1 /b 2 -KO and C57BL/6 mice which peaked at 5 days post-injury ( Fig. 4A & B). Injury-induced expression of CD68 and F4/80 was significantly higher in b 1 /b 2 -KO mice than in uninjured controls (2-way ANOVA, main strain effect).
An examination of myogenic regulatory factor (MRF) mRNA revealed that Myf5, MyoD and myogenin expression all increased after myotoxic damage, peaking at 5 days post-injury (Fig. 5A, B and C). Expression of all three MRFs was significantly higher in b 1 /b 2 -KO mice (2-way ANOVA, main strain effect), and also seemed to be more sustained than the increases in control muscles, with MyoD and myogenin in particular remaining elevated for longer than 5 days post-injury (Fig. 5A, B and 5C).

Deletion of b 1 -and b 2 -ARs increases myoblast proliferation and inhibits differentiation ex vivo
To directly determine the proliferation and differentiation of myoblasts from b 1 /b 2 -KO mice without the confounding variables inherent to the whole-body knockout mouse, we isolated and cultured myoblasts from b 1 /b 2 -KO mice and C57BL/6 controls. We found that myoblasts from b 1 /b 2 -KO mice proliferated more rapidly and differentiated less effectively than those from C57BL/ 6 controls (Fig. 6).

Discussion
Although exogenous stimulation of b-ARs has been shown to enhance mammalian muscle fiber regeneration [6][7][8][9], and recent studies have also implicated components of the b-AR signalling pathway in muscle regeneration and hypertrophy [10][11][12], the contribution of b-ARs to the muscle regenerative process per se is less well understood. The findings from the present study have addressed this gap in existing knowledge by examining the functional capacity of skeletal muscles from b 1 -, b 2 -and b 1 /b 2 -KO mice. While many previous studies have examined b-KO mice in a cardiovascular context, the attributes of skeletal muscle are less well described. [22]. We found that the mass of TA muscles from either b 1 -KO mice or b 2 -KO mice were similar to respective controls, whereas the TA muscles from b 1 /b 2 -KO mice were significantly smaller than control. Muscle fiber CSA in the various strains closely mirrored muscle mass, with neither of the single knockout strains displaying significant differences in CSA while the double knockouts had a significant reduction. Hinkle and colleagues [22] also found no difference in TA mass between b 1 -KO mice and control, but noted that both b 2 -KO mice and b 1 / b 2 -KO mice exhibited significantly reduced TA muscle mass than controls, from which they concluded that muscle size was positively regulated by b 2 -ARs but not b 1 -ARs. In the present study, we did not observe any significant difference in TA muscle mass in b 2 -KO mice, but did note a significant reduction in TA mass in b 1 /b 2 -KO mice. The reason for the discrepancy between the studies is unclear but may reflect the back-crossing onto a different background strain (C57BL/6) in the present study. There was no significant difference in TA mass for either b 1 -KO or b 2 -KO mice when compared with littermate controls, but b 1 /b 2 -KO mice had a significantly lower TA mass than control (*P,0.05 compared with control, t-test, n = 5-6 per group). (C) and (D) maximal tetanic force production by TA muscles measured as either absolute force (P o ) or force normalized to muscle size (specific force, or sP o ). b 1 -KO mice had no significant differences in maximal force whether expressed as either P o or sP o . In contrast, b 2 -KO mice had significant deficits in maximal force when expressed as either P o or sP o . b 1 /b 2 -KO mice had significantly lower maximal forces than control when expressed as P o , but there was no significant deficit in force production when force was normalized to muscle size (sP o ) (*P,0.05 compared with control, t-test, n = 5-6 per group). (E) Comparison of cross-sectional area (CSA) of TA muscle fibers from b-KO mouse strains (white bars) and their respective controls (grey bars). Neither b 1 -KO nor b 2 -KO mice had significant changes in muscle fiber CSA compared with their wild-type littermates, but fiber CSA was significantly lower in TA muscles from b 1 /b 2 -KO mice compared with control. Data for each strain were pooled from .1500 fibers from n$9 mice (*P,0.05, Mann-Whitney test by ranks).  Table 2. Selected isometric twitch contractile properties of uninjured TA muscles from b-KO mice.  The muscles of mice lacking b 1 -ARs were identical to controls for every functional parameter measured, but muscles from both b 2 -KO mice and b 1 /b 2 -KO mice produced less absolute force than controls (whether twitch force or tetanic force) and exhibited a slower rate of force generation. The only difference we observed in functionality between the muscles of b 2 -KO mice and b 1 /b 2 -KO mice was that of specific force (sP o ). When expressed this way muscles from b 1 /b 2 -KO mice produced similar forces to control, whereas force production by b 2 -KO mice remained significantly depressed relative to control muscles. Taken together, our data indicate that the loss of b 2 -ARs has profound effects on skeletal muscle morphology and contractile function, but the loss of b 1 -ARs does not significantly impact on either parameter. This is perhaps not surprising given that b 1 -ARs make up only about 7-10% of the total b-ARs in skeletal muscle [23]. It is worth noting however that the expression of b 1 -ARs is relatively higher in slowtwitch muscles such as the soleus, and that the present study examined the TA and EDL that are both predominantly fasttwitch muscles. We therefore cannot exclude the possibility that the loss of b 1 -ARs may have an impact on contractile function in slow twitch muscles such as the soleus. These findings also confirm previous studies demonstrating that b 2 -AR signaling has significant effects on force producing capacity [24,25].
In the context of muscle regeneration, we found that the muscles from b 1 -KO mice did not show any significant differences in force production after myotoxic injury compared with controls. In contrast, muscles from b 2 -KO mice displayed significant deficits in force production during regeneration when measured as either absolute force or specific force. When we accounted for the fact that the muscles of b 2 -KO mice were returning to a lower absolute force (i.e. we expressed force production as '% uninjured'), we found no significant differences in the restoration of force Table 3. Selected isometric twitch contractile properties of uninjured EDL muscles from b 1 /b 2 -KO mice and C57BL/6 controls.  production in regenerating muscles between b 2 -KO mice and controls. Similarly, muscles from b 1 /b 2 -KO (double knockout) mice exhibited significant differences in absolute force production compared with controls, but when force was normalized to either muscle cross-sectional area (sP o ) or to force production by uninjured muscles (% uninjured) there was no difference in the restoration of force over the 14 day period of regeneration. If the data are normalized to reflect the intrinsic differences in muscle strength and the entire 14 day period of regeneration is analysed in its entirety, then a lack of b-ARs (either b 1 -ARs, b 2 -ARs or both b 1 -and b 2 -ARs) does not appear to affect the restoration of muscle function after myotoxic damage. If, however, we confine our analysis to the earliest time point of the regeneration period (i.e. 7 days post-injury), a different pattern emerges. In this case, early regeneration of the TA muscle in both b 1 -KO mice and b 2 -KO is unaffected compared with controls, whereas the muscles of b 1 /b 2 -KO mice exhibit significant force deficits. Our results suggest a level of redundancy between the two subtypes of b-AR. This finding may be confounded by these mice having lifelong knockout of b-ARs, which may promote possible compensatory adaptations in b-AR signaling that accompany such models. In addition, it seems unlikely that b 1 -ARs, which comprise only 7-10% of b-ARs within muscle [23] could compensate for the loss of b 2 -ARs. Nonetheless, the possibility of b-AR redundancy requires further investigation, possibly by other experimental models such as inducible gene knock-out, or in vivo knockdown of b-ARs with siRNA, that would be less likely to be confounded by compensatory alterations in signaling.
To confirm our findings in the TA muscle, we also examined the characteristics of the EDL muscles. Since our findings from the TA muscle had indicated only b 1 /b 2 -KO mice exhibited differences in regeneration after myotoxic injury, we confined our analysis of EDL morphology and function to b 1 /b 2 -KO mice and C57BL/6 controls. Uninjured EDL muscles from b 1 /b 2 -KO mice exhibited similar differences in morphology and function to TA muscles; i.e. decreased muscle mass, decreased twitch force and rate of contraction, and decreased absolute tetanic force when compared with C57BL/6 controls. Furthermore, EDL muscles from b 1 /b 2 -KO mice displayed similar deficits in regeneration at 7 days post-injury, confirming our findings in the TA muscle and further suggesting that the loss of both b 1 -and b 2 -ARs affects the earliest period of regeneration after myotoxic damage.
Skeletal muscle regeneration is preceded by a well-defined and highly coordinated inflammatory response, involving the infiltration of neutrophils and macrophages at the site of damage and subsequent release of pro-and anti-inflammatory cytokines [26]. Non-cytokine anti-inflammatory mediators are also able to modulate the inflammatory process, including glucocorticoids, adenosine, and endogenous b-agonists [27]. b-ARs are not only present in skeletal muscle [23], but have also been detected on the surface of inflammatory cells such as mast cells, eosinophils, neutrophils and macrophages [28]. To determine the degree of macrophage infiltration in regenerating muscles, we examined the expression of the macrophage-specific markers F4/80 and CD68 [29]. The expression of both macrophage markers was significantly increased in b 1 /b 2 -KO than in controls. Previous studies have revealed that macrophages incubated with LPS have an increased expression of TNF-a and IL-6, which is inhibited by the b-agonist clenbuterol [30,31] and that administering the b-AR antagonist propranolol can potentiate the release of inflammatory cytokines in vivo [32,33]. Due to the well-characterized increase in TNF-a and IL-6 in response to inflammation, as well as the evidence that their expression is controlled to some degree by b-AR signaling, we examined these two cytokines as a measure of inflammation and found that both cytokines were increased acutely after injury, with maximal expression at 2 days post-injury in control mice. This correlates well with previous studies from our laboratory where we have reported that oedema and immune cell infiltration are maximal at this time [15]. In the present study, TNF-a was significantly more elevated in b 1 /b 2 -KO mice, and IL-6 mRNA showed a non-significant trend toward higher expression than in regenerating muscles from control mice, suggesting that the acute immune response to injury in b 1 /b 2 -KO mice is exacerbated by the lack of b-ARs. Taken together, these data suggest that the inflammatory response and subsequent macrophage infiltration after injury is acutely higher in the b 1 /b 2 -KO mice compared with control.
To ascertain whether the observed force deficits in muscles from b 1 /b 2 -KO mice at 7 days post-injury were associated with impaired myofiber regeneration, we assessed the expression of the myogenic regulatory factors (MRFs) responsible for muscle formation. Previous studies have demonstrated that clenbuterol represses the expression of MyoD and myogenin in denervated rat soleus muscles [34], and that clenbuterol administration increased myogenin expression in immobilized rat plantaris muscles [35]. Clenbuterol administration also increased MyoD expression in rat soleus muscles [36]. In the present study, Myf5 (the transcription factor responsible for specification of satellite cells to the myogenic lineage) was upregulated after myotoxin injection, with the increase in expression peaking at 5 days post-injury. Furthermore, expression of Myf5 was significantly increased in the regenerating muscles of b 1 /b 2 -KO mice than controls, indicating that myoblast proliferation was not compromised in the b 1 /b 2 -KO mice, and may be propagated for longer after muscle injury, than in control mice. The induction of MyoD (which initiates the terminal differentiation program in myoblasts) and myogenin (which is involved in the activation of muscle-specific genes in the immature muscle cells [37]) following injury was also exaggerated in the muscles of b 1 /b 2 double-KO mice. Taken together, our MRF expression data suggest that myoblast proliferation and differentiation may be enhanced in b 1 /b 2 -KO mice at the expense of moderately delayed differentiation. This observation, consistent with what we had expected and described in a previous review [38], may explain why force producing capacity is impaired at 7 days post-injury in b 1 /b 2 -KO mice, but that muscles are capable of restoring functionality (relative to uninjured muscles) similar to control animals at 10 days post-injury. This rapid 'catch up' where the muscles of b 1 /b 2 -KO mice seemingly overcome their initial delayed regeneration and function impairment, is supported by our observations of cultured primary myoblasts from b 1 /b 2 -KO mice, where proliferation was enhanced and prolonged and differentiation was delayed.
The present study utilized whole body b 1 /b 2 -KO mice, since to our knowledge there are no muscle-specific b 1 /b 2 -KO mice currently available. One concern with using the whole body b 1 / b 2 -KO mouse (a concern that is also valid in any in vivo study of b-agonist administration) is that any effects on muscle regeneration may be a consequence of perturbations of non-muscle physiological systems, rather than a direct effect on muscle regeneration per se. For example, the altered inflammatory response observed in the present study, while not a direct result of the muscle lacking b-ARs, undoubtedly influenced fiber regeneration.
To obviate these concerns we isolated myoblasts from both b 1 / b 2 -KO mice and C57BL/6 controls to examine myoblast proliferation and differentiation in the absence of confounding factors, and found that myoblasts isolated from b 1 /b 2 -KO mice proliferated more rapidly and differentiated far less effectively than those from C57BL/6 controls. While initially this may seem to be at odds with our MRF expression data from regenerating muscles, it must be remembered that even if myoblast differentiation was impaired in vivo, the vastly greater number of myoblasts present in the muscle due to the increased proliferation would still result in an overall increase in MyoD and myogenin expression in the muscle. Interestingly, we have previously documented a dramatic increase in the gene expression of adrb1 and adrb2 (b 1 -and b 2adrenoceptors, respectively) during the switch from proliferation to differentiation [38]. Combined with the findings of the present study, these data support a role for b 1 /b 2 -ARs in inhibiting myoblast proliferation and promoting differentiation.
Another concern with whole body b 1 /b 2 -KO mice is the potential for cardiovascular disturbances (particularly alterations in local blood flow) to influence muscle regeneration. We do not believe that the muscles from b 1 /b 2 -KO mice suffered a significant deficit in perfusion as this would have resulted in a constant inhibition of regeneration, whereas we observed a deficit in regeneration only at 7 days post-injury. In fact, the muscles from b 1 /b 2 -KO mice subsequently regenerated faster than control in order to 'catch up' to the control muscles at the later time points.
With all our data taken together we propose a model in which b 1 /b 2 -KO mice exhibit an enhanced inflammatory response to injury and enhanced myoblast proliferation during regeneration. We believe that b 1 /b 2 -KO mice have delayed early regeneration due to the prolonged and enhanced inflammatory response, but once the inflammation subsides, the enhanced myoblast proliferation allows the b 1 /b 2 -KO muscles to regenerate rapidly -thus accounting for the rapid 'catch up' of muscles between 7 and 10 days post-injury. Further studies using muscle specific knockdown of b-ARs or virally-mediated 'knock in' of b-ARs to inflammatory cells of b 1 /b 2 -KO mice, or other injury models (such as crush or burn injuries) with a higher inflammatory component would help test this hypothesis.
In summary, our findings indicate that b-ARs play an important role in early muscle regeneration, at least in part via a direct effect on myoblast proliferation and differentiation. Manipulation of b-AR signaling during these early stages of regeneration may therefore improve the rate, extent and efficacy of the regenerative process, to enhance functional recovery after injury. Figure S1 Maximal tetanic force of regenerating TA muscles of different strains of b-KO mice following injury and expressed as absolute force (P o ). Force production by b 1 -KO mice did not differ from controls during regeneration (A), but both b 2 -KO mice (B) and b 1 /b 2 -KO mice (C) had significant force deficits during all stages of regeneration (#P,0.05, strain main effect, 2-way ANOVA). (TIF)